Compositions and therapeutic methods using morphogenic proteins, hormones and hormone receptors

ABSTRACT

This invention features devices and methods for inducing tissue formation in a mammal, involving the use of a morphogenic protein, a hormone and a soluble receptor of the hormone. The hormone and receptor thereof are used to enhance the tissue inductive activity of the morphogenic protein.

CROSS REFERENCE TO RELATED APPLICATION

[0001] Under 35 U.S.C. § 119(e)(1), this application claims the benefitof prior U.S. provisional application serial No. 60/156,261, filed Sep.27, 1999.

BACKGROUND OF THE INVENTION

[0002] The Transforming Growth Factor-Beta (“TGF-β”) superfamilyrepresents a large number of evolutionarily conserved morphogenicproteins with diverse activities in growth, differentiation, tissuemorphogenesis and repair. This superfamily includes osteogenic proteins(“OPs”) and bone morphogenic proteins (“BMPs”). OPs and BMPs share ahighly conserved, bioactive cysteine-rich domain near their C-terminiand have a propensity to form homo- and hetero-dimers.

[0003] Many morphogenic proteins belonging to the BMP family have beendescribed. Some were isolated using purification techniques on the basisof osteogenic activity. Others were identified and cloned by virtue ofDNA sequence homologies within conserved regions that are common to theBMP family. These homologs are referred to as consecutively numberedBMPs whether or not they have demonstrable osteogenic activity. Whileseveral of the earliest members of the BMP family were identified byvirtue of their ability to induce new cartilage and bone, a number ofother BMPs have different or additional tissue-inductive capabilities.For example, BMP-12 and BMP-13 (identified by DNA sequence homology)reportedly induce tendon/ligament-like tissue formation in vivo (WO95/16035). Several BMPs, including some of those originally isolated onthe basis of their osteogenic activity, can induce neuron proliferationand promote axon regeneration (WO 95/05846; Liem et al., Cell, 82, pp.969-79 (1995)). Thus, it appears that BMPs may have a variety ofpotential tissue-inductive capabilities whose final expression dependson a complex set of developmental and environmental cues.

[0004] Many of the mammalian BMPs have been recombinantly expressed asactive bomo- or heterodimers in a variety of host systems, makingtherapeutic treatments using morphogenic proteins feasible. Implantableosteogenic devices comprising mammalian osteogenic protein for promotingbone healing and regeneration have been described (see, e.g., Oppermannet al., U.S. Pat. No. 5,354,557). Some osteogenic devices containporous, biocompatible matrices that allow the diffusion of osteogenicproteins into the implantation site as well as the influx and efflux ofprogenitor cells. Osteogenic protein-coated prosthetic devices thatenhance the bond strength between the prosthesis and existing bone havealso been described (Rueger et al., U.S. Pat. No. 5,344,654).

SUMMARY OF THE INVENTION

[0005] This invention is based on the discovery that thetissue-inductive activity of a morphogenic protein can be enhanced by ahormone in the presence of a soluble receptor of the hormone.

[0006] Accordingly, this invention features a method for improving thetissue inductive capability of a morphogenic protein at a target locusin a mammal. In this method, the morphogenic protein and a firsteffective amount of a hormone and a second effective amount of a solublereceptor of the hormone are administered to the target locus, whereinthe morphogenic protein is capable of inducing tissue formation whenaccessible to a progenitor cell in the mammal, and the hormone and thereceptor in combination enhance that capability. The morphogenicprotein, hormone and hormone receptor can be administered simultaneouslyto the target locus. Alternatively, the three components areadministered separately, in any order: for instance, the morphogenicprotein can be administered first, and then the hormone and hormonereceptor are administered together; or the morphogenic protein and thehormone are administered together first, and then the hormone receptoris administered. In one embodiment, the morphogenic protein isadministered via a nucleic acid (e.g., a plasmid, a viral vector, ornaked DNA) that comprises a sequence encoding the morphogenic proteinand is capable of expressing the morphogenic protein in the appropriateprogenitor cells of a patient.

[0007] The morphogenic protein may comprise a pair of subunitsdisulfide-bonded to produce a dimeric species, wherein at least one ofthe subunits comprises a polypeptide belonging to the BMP proteinfamily. For instance, the morphogenic protein may comprise an amino acidsequence sufficiently duplicative of the amino acid sequence of areference BMP such as BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7 (OP-1),BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, COP-5, or COP-7, such thatit has morphogenic activity similar to that of the reference BMP. In onepreferred embodiment, the morphogenic protein is a homo- or heterodimercomprising a BMP-2 or BMP-7 (OP-1) subunit.

[0008] The morphogenic protein is capable of inducing tissue formation.For instance, it may be capable of inducing the progenitor cell to formtissue tendon/ligament-like or neural-like tissue; or it may be anosteogenic protein that is capable of inducing the progenitor cell toform endochondral or intramembranous bone, or cartilage. The method ofthis invention thus can be used to induce tissue regeneration or repairin a variety of tissue defects such as bone, cartilage, soft tissue andneural tissue defects.

[0009] Hormones useful in this invention include but are not limited tocytokines (e.g., interleukins 1 through 18), growth factors (e.g.,fibroblast growth factor, vascular endothelial growth factor,platelet-derived growth factor, TGF-β, or prostaglandin) or morphogenicproteins.

[0010] The invention also features pharmaceutical compositions and kitscomprising a hormone and a soluble receptor thereof for improving thetissue inductive activity of a morphogenic protein. This invention alsoprovides implantable morphogenic devices for inducing tissue formationin allogeneic and xenogeneic implants. Such devices comprise amorphogenic protein, a hormone and a soluble receptor thereof disposedwithin a carrier. Methods for inducing local tissue formation from aprogenitor cell in a mammal using those compositions and devices arealso provided. A method for accelerating allograft repair in a mammalusing those morphogenic devices is provided. This invention alsoprovides a prosthetic device comprising a prosthesis coated with amorphogenic protein, a hormone and a soluble receptor thereof, and amethod for promoting in vivo integration of an implantable prostheticdevice to enhance the bond strength between the prosthesis and theexisting target tissue at the joining site. Methods for treating tissuedegenerative conditions in a mammal using the pharmaceuticalcompositions are also provided.

[0011] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intended to be limiting.

[0012] Other features and advantages of the invention will be apparentfrom the following drawings, detailed description, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a bar graph showing that a combination of interleukin 6(“IL-6”) and soluble IL-6 receptor (“sIL-6R”) significantly increasesthe ability of OP-1 to induce alkaline phosphatase (“AP”) activity infetal rat calvaria (“FRC”) cells. “OP” stands for “OP-1”; “IL-6R” refersto “sIL-6R.” The parenthesized numbers indicate the proteinconcentrations (ng/ml) used in the assay; in the case of IL-6/sIL-6Rcombinations, the two numbers separated by a backslash in theparenthesis indicate the protein concentrations of IL-6 and sIL-6R,respectively.

[0014]FIG. 2 is a photograph showing results of a mineralized bonenodule formation assay using OP-1 and IL-6. Dark spots inside the wellsrepresent mineralized bone nodules.

[0015]FIG. 3 is a photograph showing results of a mineralized bonenodule formation assay using OP-1, IL-6 and sIL-6R. Dark spots insidethe wells represent mineralized bone nodules.

[0016]FIG. 4 is a bar graph showing the mRNA levels of BMPR-IA, BMPR-IB,ActR-I, and BMPR-II in various test groups. “sR” stands for sIL-6R.Values in the graph represent the means±SE of twelve Northern blots withRNA isolated from two different FRC cell preparations.

[0017]FIG. 5 is a bar graph showing that the AP activity in FRC cellstransfected with the OP-1-encoding pW24 plasmid is enhanced by exogenoussIL-6R alone or a combination of IL-6 and sIL-6R (“IL-6/R”). “IL6R”stands for sIL-6R. Values in the graph represent the mean±SE of fiveindependent determinations with three different FRC cell preparationsand two different DNA preparations. “IL-6/R (X/Y)” refers to X ng/mlIL-6 and Y ng/ml sIL-6R.

DETAILED DESCRIPTION OF THE INVENTION

[0018] In order that the invention herein described may be fullyunderstood, the following detailed description is set forth.

[0019] The term “biocompatible” refers to a material that does notelicit detrimental effects associated with the body's various protectivesystems, such as cell and humoral-associated immune responses, e.g.,inflammatory responses and foreign body fibrotic responses. This termalso implies that no specific undesirable effects, cytotoxic orsystemic, are caused by the material when it is implanted into thepatient.

[0020] The term “BMP” refers to a protein belonging to the BMP family ofthe TGF-β superfamily of proteins defined on the basis of DNA and aminoacid sequence homology. According to this invention, a protein belongsto the BMP family when it has at least 50% (e.g., at least 70% or even85%) amino acid sequence homology with a known BMP family member withinthe conserved C-terminal cysteine-rich domain that characterizes the BMPfamily. Members of the BMP family may have less than 50% DNA or aminoacid sequence homology overall.

[0021] The term “morphogenic protein” refers to a protein havingmorphogenic activity. For instance, this protein is capable of inducingprogenitor cells to proliferate and/or to initiate differentiationpathways that lead to the formation of cartilage, bone, tendon,ligament, neural or other types of tissue, depending on localenvironmental cues. Thus, morphogenic proteins useful in this inventionmay behave differently in different surroundings. A morphogenic proteinof this invention may comprise at least one polypeptide belonging to theBMP family.

[0022] The term “osteogenic protein” refers to a morphogenic proteinthat is capable of inducing a progenitor cell to form cartilage and/orbone. The bone may be intramembranous bone or endochondral bone. Mostosteogenic proteins are members of the BMP family and are thus alsoBMPs. However, the converse may not be true. According to thisinvention, a BMP identified by sequence homology must have demonstrableosteogenic or chondrogenic activity in a functional bioassay to be anosteogenic protein.

[0023] The terms “morphogenic activity,” “inducing activity” and “tissueinductive activity” alternatively refer to the ability of an agent tostimulate a target cell to undergo one or more cell divisions(proliferation) that may optionally lead to cell differentiation. Suchtarget cells are referred to generically herein as progenitor cells.Cell proliferation is typically characterized by changes in cell cycleregulation and may be detected by a number of means which includemeasuring DNA synthetic or cellular growth rates. Early stages of celldifferentiation are typically characterized by changes in geneexpression patterns relative to those of the progenitor cell; suchchanges may be indicative of a commitment towards a particular cell fateor cell type. Later stages of cell differentiation may be characterizedby changes in gene expression patterns, cell physiology and morphology.Any reproducible change in gene expression, cell physiology ormorphology may be used to assess the initiation and extent of celldifferentiation induced by a morphogenic protein.

[0024] The term “synergistic interaction” refers to an interaction inwhich the combined effect of two or more agents is greater than thealgebraic sum of their individual effects.

[0025] The term “hormone/receptor pair” refers to a combination of ahormone and a soluble receptor thereof. The hormone (e.g., a cytokine, agrowth factor, or a morphogenic protein) can be of any mammalian origin(e.g., human, bovine, or murine). A soluble receptor of a hormone is acompound that binds specifically to the hormone, and can, for example,be a polypeptide containing only the hormone-binding domain (e.g., anextracellular domain) of a native cellular receptor of the hormone, anantibody specific for the hormone, or a chemical compound thatspecifically interacts with the hormone. A soluble receptor can also bea compound (e.g., a protein) containing a domain that specifically bindsto the hormone and another domain that specifically binds to the nativecellular receptor of the hormone such that the soluble receptorfacilitates the binding of the hormone to its native cellular receptor;an example of such a soluble receptor is the IGF-binding protein.

[0026] Morphogenic Proteins

[0027] The morphogenic proteins of this invention are capable ofstimulating a progenitor cell to undergo cell division and/ordifferentiation. They may belong to the TGF-13 protein superfamily, andinclude, but are not limited to, OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-3b,BMP-4, BMP-5, BMP-6, BMP-9, BMP-10; BMP-11, BMP-12, BMP-13, BMP-14,BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10,GDF-11, GDF-12, DPP, Vg-1, Vgr-1, 60A protein, NODAL, UNIVIN, SCREW,ADMP, NEURAL, and TGF-β.

[0028] One of the preferred morphogenic proteins is OP-1. Nucleotide andamino acid sequences for hOP-1 are provided in SEQ ID NOs:1 and 2,respectively. For ease of description, hOP-1 is recited as arepresentative morphogenic protein. It will be appreciated by theordinarily skilled artisan that OP-1 is merely representative of afamily of morphogens.

[0029] Other useful morphogenic proteins also include polypeptideshaving at least 50% (e.g., at least 70% or even 85%) sequence homologywith a known morphogenic protein, particularly with a known BMP withinthe conserved C-terminal cysteine-rich domain that characterizes the BMPprotein family. These morphogenic proteins include biologically activevariants of any known morphogenic protein, including variants containingconservative amino acid changes. For instance, useful morphogenicproteins include those containing sequences that share at least 70%amino acid sequence homology with the C-terminal seven-cysteine domainof hOP-1, which domain corresponds to the C-terminal 102-106 amino acidresidues of SEQ ID NO:2. The C-terminal 102 amino acid residuescorresponds to residues 330-431 of SEQ ID NO:2. In one embodiment ofthis invention, the morphogenic protein consists of a pair of subunitsdisulfide-bonded to produce a dimer, wherein at least one of thesubunits comprises a recombinant polypeptide belonging to the BMPfamily.

[0030] As used herein, “amino acid sequence homology” is understood toinclude both amino acid sequence identity and similarity. Homologoussequences share identical and/or similar amino acid residues, wheresimilar residues are conservative substitutions for, or “allowed pointmutations” of, corresponding amino acid residues in an aligned referencesequence. Thus, a candidate polypeptide sequence that shares 70% aminoacid homology with a reference sequence is one in which any 70% of thealigned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence.Certain particularly preferred morphogenic polypeptides share at least60% (e.g., at least 65%) amino acid sequence identity with theC-terminal seven-cysteine domain of human OP-1.

[0031] As used herein, “conservative substitutions” are residues thatare physically or functionally similar to the corresponding referenceresidues. That is, a conservative substitution and its reference residuehave similar size, shape, electric charge, chemical properties includingthe ability to form covalent or hydrogen bonds, or the like. Preferredconservative substitutions are those fulfilling the criteria defined foran accepted point mutation in Dayhoff et al., Atlas of Protein Sequenceand Structure 5:345-352 (1978 & Supp.). Examples of conservativesubstitutions are substitutions within the following groups: (a) valine,glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d)aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine,threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,tyrosine. The term “conservative variant” or “conservative variation”also includes the use of a substituting amino acid residue in place ofan amino acid residue in a given parent amino acid sequence, whereantibodies specific for the parent sequence are also specific for, i.e.,“cross-react” or “immuno-react” with, the resulting substitutedpolypeptide sequence.

[0032] Amino acid sequence homology can be determined by methods wellknown in the art. For instance, to determine the percent homology of acandidate amino acid sequence to the sequence of the seven-cysteinedomain, the two sequences are first aligned. The alignment can be madewith, e.g., the dynamic programming algorithm described in Needleman etal., J. Mol. Biol. 48:443 (1970), and the Align Program, a commercialsoftware package produced by DNAstar, Inc. The teachings by both sourcesare incorporated by reference herein. An initial alignment can berefined by comparison to a multi-sequence alignment of a family ofrelated proteins. Once the alignment is made and refined, a percenthomology score is calculated. The aligned amino acid residues of the twosequences are compared sequentially for their similarity to each other.Similarity factors include similar size, shape and electrical charge.One particularly preferred method of determining amino acid similaritiesis the PAM250 matrix described in Dayhoff et al., supra. A similarityscore is first calculated as the sum of the aligned pairwise amino acidsimilarity scores. Insertions and deletions are ignored for the purposesof percent homology and identity. Accordingly, gap penalties are notused in this calculation. The raw score is then normalized by dividingit by the geometric mean of the scores of the candidate sequence and theseven-cysteine domain. The geometric mean is the square root of theproduct of these scores. The normalized raw score is the percenthomology.

[0033] Morphogenic proteins useful herein include any known naturallyoccurring native proteins, including allelic, phylogenetic counterpartsand other variants thereof. These variants include forms having varyingglycosylation patterns, varying N-termini, and active truncated ormutated forms of a native protein. Useful morphogenic proteins alsoinclude those that are biosynthetically produced (e.g., “muteins” or“mutant proteins”) and those that are new, morphogenically activemembers of the general morphogenic family of proteins. Particularlyuseful sequences include those comprising the C-terminal 96 to 102 aminoacid residues of: DPP (from Drosophila), Vg-1 (from Xenopus), Vgr-1(from mouse), the OP1 and OP2 proteins (U.S. Pat. No. 5,011,691), aswell as the proteins referred to as BMP-2, BMP-3, BMP-4 (WO 88/00205,U.S. Pat. No. 5,013,649 and WO 91/18098), BMP-5 and BMP-6 (WO 90/11366),BMP-8 and BMP-9. Other proteins useful in the practice of the inventioninclude active forms of OP1, OP2, OP3, BMP-2, BMP-3, BMP-3b, BMP-4,BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15,DPP, Vg-1, Vgr-1, 60A protein, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7,GDF-8, GDF-9, and GDF-10, GDF-11, GDF-12, GDF-13, UNIVIN, NODAL, SCREW,ADMP, NEURAL, and TGF-β.

[0034] Osteogenic proteins useful as morphogenic proteins of thisinvention include those containing sequences that share greater than 60%identity with the seven-cysteine domain. In other embodiments, usefulosteogenic proteins are defined as osteogenically active proteins havingany one of the generic sequences defined herein, including OPX (SEQ IDNO:3) and Generic Sequences 7 (SEQ ID NO:4), 8 (SEQ ID NO:5), 9 (SEQ IDNO:6) and 10 (SEQ ID NO:7).

[0035] Generic Sequence 7 (SEQ ID NO:4) and Generic Sequence 8 (SEQ IDNO:5), disclosed below, accommodate the homologies shared amongpreferred protein family members identified to date, including OP-1,OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, 60A, DPP, Vg-1, Vgr-1,and GDF-1. The amino acid sequences for these proteins are describedherein and/or in the art. The generic sequences include the identicalamino acid residues shared by these sequences in the C-terminal six- orseven-cysteine skeletal domains (represented by Generic Sequences 7 and8, respectively), as well as alternative residues for the variablepositions within the sequences. The generic sequences provide anappropriate cysteine skeleton where inter- or intra-molecular disulfidebonds can form. Those sequences contain certain specified amino acidsthat may influence the tertiary structure of the folded proteins. Inaddition, the generic sequences allow for an additional cysteine atposition 36 (Generic Sequence 7) or position 41 (Generic Sequence 8),thereby encompassing the biologically active sequences of OP-2 and OP-3.GENERIC SEQUENCE 7           Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp XaaXaa Xaa Xaa Xaa Xaa (SEQ ID NO:4)           1                  5                   10                    15 Pro Xaa Xaa Xaa Xaa Ala XaaTyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro Xaa Xaa              20                 25                    30 Xaa Xaa Xaa Xaa Xaa Xaa AsnHis Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa35                    40                   45                  50 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa         55                   60                  65                   70Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa                 75                         80                  85 MetXaa Val Xaa Xaa Cys Xaa Cys Xaa       90                   95

[0036] wherein each Xaa is independently defined as follows (“Res.”means “residue”): Xaa at res.2=(Tyr or Lys); Xaa at res.3=(Val or Ile);Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys orAla); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu, Val or Ile); Xaa atres.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.12=(Asp, Arg, Asn orGlu); Xaa at res. 13=(Trp or Ser); Xaa at res.14=(Ile or Val); Xaa atres.15.=(Ile or Val); Xaa at res. 16 (Ala or Ser); Xaa at res. 18=(Glu,Gln, Leu, Lys, Pro or Arg); Xaa at res. 19=(Gly or Ser); Xaa at res.20(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Gln, Leu or Gly);Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr, Asp, Gln,Ala or Ser); Xaa at res.28=(Glu, Lys, Asp, Gln or Ala); Xaa atres.30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa at res.31=(Phe, Leu orTyr); Xaa at res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp, Ala, Thror Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa atres.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly orLeu); Xaa at res.38=(Asn, Ser or Lys); Xaa at res.39=(Ala, Ser, Gly orPro); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile, Val or Thr);Xaa at res.45=(Val, Leu, Met or Ile); Xaa at res.46=(Gln or Arg); Xaa atres.47=(Thr, Ala or Ser); Xaa at res.48=(Leu or Ile); Xaa at res.49=(Valor Met); Xaa at res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn,Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val, Gly or Leu);Xaa at res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Seror Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa atres.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa atres.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa atres.59=(Lys, Leu or Glu); Xaa at res.60=(Pro, Val or Ala); Xaa atres.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa atres.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa atres.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser); Xaa atres.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa atres.72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa atres.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa atres.77=(Asp, Glu, Asn, Arg or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr orAsp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thror Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg);Xaa at res.85=(Lys, Asn, Gln, His, Arg or Val); Xaa at res.86=(Tyr, Gluor His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu,Trp or Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg,Lys, Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa atres.95=(Gly or Ala); and Xaa at res.97=(His or Arg).

[0037] Generic Sequence 8 (SEQ ID NO:5) includes all of Generic Sequence7 and in addition includes the following five amino acid at itsN-terminus: Cys Xaa Xaa Xaa Xaa (SEQ ID NO:8), wherein Xaa atres.2=(Lys, Arg, Ala or Gln); Xaa at res.3=(Lys, Arg or Met); Xaa atres.4=(His, Arg or Gln); and Xaa at res.5=(Glu, Ser, His, Gly, Arg, Pro,Thr, or Tyr). Accordingly, beginning with residue 7, each “Xaa” inGeneric Sequence 8 is a specified amino acid as defined as for GenericSequence 7, with the distinction that each residue number described forGeneric Sequence 7 is shifted by five in Generic Sequence 8. Forexample, “Xaa at res.2=(Tyr or Lys)” in Generic Sequence 7 correspondsto Xaa at res.7 in Generic Sequence 8.

[0038] Generic Sequences 9 (SEQ ID NO:6) and 10 (SEQ ID NO:7) arecomposite amino acid sequences of the following proteins: human OP-1(“hOP-1”), hOP-2, hOP-3, hBMP-2, hBMP-3, hBMP-4, hBMP-5, hBMP-6, hBMP-9,hBMP10, hBMP-11, Drosophila 60A, Xenopus Vg-1, sea urchin UNIVIN,hCDMP-1 (mouse GDF-5 or “mGDF-5”), hCDMP-2 (mGDF-6, hBMP-13), hCDMP-3(mGDF-7, hBMP-12), mGDF-3, hGDF-1, mGDF-1, chicken DORSALIN, DPP,Drosophila SCREW, mouse NODAL, mGDF-8, hGDF-8, mGDF-9, mGDF-10, hGDF-11,mGDF-11, hBMP-15, and rat BMP3b. Like Generic Sequence 7, GenericSequence 9 accommodates the C-terminal six-cysteine skeleton and, likeGeneric Sequence 8, Generic Sequence 10 accommodates the C-terminalseven-cysteine skeleton. GENERIC SEQUENCE 9        Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa (SEQ ID NO:6)       1                 5                    10                   15Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Xaa            20                   25                   30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa     35                   40                   45                   50Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa                 55                   60                        65 XaaXaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa         70                  75                    80 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa85                   90                    95

[0039] wherein each Xaa is independently defined as follows: Xaa atres.1=(Phe, Leu or Glu); Xaa at res.2=(Tyr, Phe, His, Arg, Thr, Lys,Gln, Val or Glu); Xaa at res.3=(Val, Ile, Leu or Asp); Xaa atres.4=(Ser, Asp, Glu, Asn or Phe); Xaa at res.5=(Phe or Glu); Xaa atres.6=(Arg, Gln, Lys, Ser, Glu, Ala or Asn); Xaa at res.7=(Asp, Glu,Leu, Ala or Gln); Xaa at res.8=(Leu, Val, Met, Ile or Phe); Xaa atres.9=(Gly, His or Lys); Xaa at res.10=(Trp or Met); Xaa at res.11=(Gln, Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at res.12=(Asp, Asn,Ser, Lys, Arg, Glu or His); Xaa at res.13=(Trp or Ser); Xaa atres.14=(Ile or Val); Xaa at res. 15=(Ile or Val); Xaa at res. 16=(Ala,Ser, Tyr or Trp); Xaa at res. 18=(Glu, Lys, Gln, Met, Pro, Leu, Arg, Hisor Lys); Xaa at res. 19=(Gly, Glu, Asp, Lys, Ser, Gln, Arg or Phe); Xaaat res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Gly, Met, Gln, His,Glu, Asp, Leu, Asn, Lys or Thr); Xaa at res.22=(Ala or Pro); Xaa atres.23=(Tyr, Phe, Asn, Ala or Arg); Xaa at res.24=(Tyr, His, Glu, Phe orArg); Xaa at res.26=(Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gln orGly); Xaa at res.28.=(Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gln);Xaa at res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp, Phe, Gln or Leu); Xaaat res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at res.32=(Pro, Ser, Alaor Val); Xaa at res.33=(Leu, Met, Glu, Phe or Val); Xaa at res.34=(Asn,Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at res.35=(Ser, Ala, Glu, Asp,Thr, Leu, Lys, Gln or His); Xaa at res.36=(Tyr, His, Cys, Ile, Arg, Asp,Asn, Lys, Ser, Glu or Gly); Xaa at res.37=(Met, Leu, Phe, Val, Gly orTyr); Xaa at res.38=(Asn, Glu, Thr, Pro, Lys, His, Gly, Met, Val orArg); Xaa at res.39=(Ala, Ser, Gly, Pro or Phe); Xaa at res.40=(Thr,Ser, Leu, Pro, His or Met); Xaa at res.41=(Asn, Lys, Val, Thr or Gln);Xaa at res.42=(His, Tyr or Lys); Xaa at res.43=(Ala, Thr, Leu or Tyr);Xaa at res.44=(Ile, Thr, Val, Phe, Tyr, Met or Pro); Xaa at res.45=(Val,Leu, Met, Ile or His); Xaa at res.46=(Gln, Arg or Thr); Xaa atres.47=(Thr, Ser, Ala, Asn or His); Xaa at res.48=(Leu, Asn or Ile); Xaaat res.49=(Val, Met, Leu, Pro or Ile); Xaa at res.50=(His, Asn, Arg,Lys, Tyr or Gln); Xaa at res.51=(Phe, Leu, Ser, Asn, Met, Ala, Arg, Glu,Gly or Gln); Xaa at res.52=(Ile, Met, Leu, Val, Lys, Gln, Ala or Tyr);Xaa at res.53=(Asn, Phe, Lys, Glu, Asp, Ala, Gin, Gly, Leu or Val); Xaaat res.54=(Pro, Asn, Ser, Val or Asp); Xaa at res.55=(Glu, Asp, Asn,Lys, Arg, Ser, Gly, Thr, Gln, Pro or His); Xaa at res.56=(Thr, His, Tyr,Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaa at res.57=(Val, Ile, Thr, Ala,Leu or Ser); Xaa at res.58=(Pro, Gly, Ser, Asp or Ala); Xaa atres.59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg or Gly); Xaa at res.60=(Pro,Ala, Val, Thr or Ser); Xaa at res.61=(Cys, Val or Ser); Xaa atres.63=(Ala, Val or Thr); Xaa at res.65=(Thr, Ala, Glu, Val, Gly, Asp orTyr); Xaa at res.66=(Gln, Lys, Glu, Arg or Val); Xaa at res.67=(Leu,Met, Thr or Tyr); Xaa at res.68=(Asn, Ser, Gly, Thr, Asp, Glu, Lys orVal); Xaa at res.69=(Ala, Pro, Gly or. Ser); Xaa at res.70=(Ile, Thr,Leu or Val); Xaa at res.71=(Ser, Pro, Ala, Thr, Asn or Gly); Xaa atres.72=(Val, Ile, Leu or Met); Xaa at res.74=(Tyr, Phe, Arg, Thr, Tyr orMet); Xaa at res.75=(Phe, Tyr, His, Leu, Ile, Lys, Gln or Val); Xaa atres.76=(Asp, Leu, Asn or Glu); Xaa at res.77=(Asp, Ser, Arg, Asn, Glu.,Ala, Lys, Gly or Pro); Xaa at res.78=(Ser, Asn, Asp, Tyr, Ala, Gly, Gln,Met, Glu, Asn or Lys); Xaa at res.79=(Ser, Asn, Glu, Asp, Val, Lys, Gly,Gln or Arg); Xaa at res.80=(Asn, Lys, Thr, Pro, Val, Ile, Arg, Ser orGln); Xaa at res.81=(Val, Ile, Thr or Ala); Xaa at res.82=(Ile, Asn,Val, Leu, Tyr, Asp or Ala); Xaa at res.83=(Leu, Tyr, Lys or Ile); Xaa atres.84=(Lys, Arg, Asn, Tyr, Phe, Thr, Glu or Gly); Xaa at res.85=(Lys,Arg, His, Gln, Asn, Glu or Val); Xaa at res.86=(Tyr, His, Glu or Ile);Xaa at res.87=(Arg, Glu, Gln, Pro or Lys); Xaa at res.88=(Asn, Asp, Ala,Glu, Gly or Lys); Xaa at res.89=(Met or Ala); Xaa at res.90=(Val, Ile,Ala, Thr, Ser or Lys); Xaa at res.91=(Val or Ala); Xaa at res.92=(Arg,Lys, Gln, Asp, Glu, Val, Ala, Ser or Thr); Xaa at res.93=(Ala, Ser, Glu,Gly, Arg or Thr); Xaa at res.95=(Gly, Ala or Thr); and Xaa atres.97=(His, Arg, Gly, Leu or Ser). Further, after res.53 in rat BMP3band mGDF-10 there is an Ile; after res.54 in GDF-I there is a Thr; afterres.54 in BMP3 there is a Val; after res.78 in BMP-8 and DORSALIN thereis a Gly; after res.37 in hGDF-1 there are Pro, Gly, Gly, and Pro.

[0040] Generic Sequence 10 (SEQ ID NO:7) includes all of GenericSequence 9 and in addition includes the following five amino acidresidues at its N-terminus: Cys Xaa Xaa Xaa Xaa (SEQ ID NO:9), whereinXaa at res.2=(Lys, Arg, Gin, Ser, His, Glu, Ala, or Cys); Xaa atres.3=(Lys, Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at res.4=(His,Gin, Arg, Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at res.5=(Gin, Thr,His, Arg, Pro, Ser, Ala, Gin, Asn, Tyr, Lys, Asp, or Leu). Accordingly,beginning at res.6, each “Xaa” in Generic Sequence 10 is a specifiedamino acid defined as for Generic Sequence 9, with the distinction thateach residue number described for Generic Sequence 9 is shifted by fivein. Generic Sequence 10. For example, “Xaa at res.1=(Phe, Leu or Glu)”in Generic Sequence 9 corresponds to Xaa at res.6 in Generic Sequence10.

[0041] As noted above, certain preferred bone morphogenic proteinsuseful in this invention have greater than 60%, preferably greater than65%, identity with the C-terminal seven-cysteine domain of hOP-1. Theseparticularly preferred sequences include allelic and phylogeneticvariants of the OP-1 and OP-2 proteins, including the Drosophila 60Aprotein. Accordingly, in certain particularly preferred embodiments,useful proteins include active proteins comprising dimers having thegeneric amino acid sequence “OPX” (SEQ ID NO:3), which defines theseven-cysteine skeleton and accommodates the homologies between severalidentified variants of OP-1 and OP-2. Each Xaa in OPX is independentlyselected from the residues occurring at the corresponding position inthe C-terminal sequence of mouse or human OP-1 or OP-2. OPX Cys Xaa XaaHis Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp Xaa Asp Trp (SEQ IDNO:3) 1                5                    10                  15 XaaIle Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe    20                 25                  30                  35 ProLeu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala Ile Xaa Gln Xaa Leu Val        40                   45                   50                    55His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu             60                  65                   70 Xaa Ala Xaa SerVal Leu Tyr Xaa Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys75                   80                   85                    90 XaaArg Asn Met Val Val Xaa Ala Cys Gly Cys His         95                   100

[0042] wherein Xaa at res.2=(Lys or Arg); Xaa at res.3=(Lys or Arg); Xaaat res.11=(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa at res. 19=(Ileor Val); Xaa at res.23=(Glu or Gln); Xaa at res.26=(Ala or Ser); Xaa atres.35=(Ala or Ser); Xaa at res.39=(Asn or Asp); Xaa at res.41=(Tyr orCys); Xaa at res.50=(Val or Leu); Xaa at res.52=(Ser or Thr); Xaa atres.56=(Phe or Leu); Xaa at res.57=(Ile or Met); Xaa at res.58=(Asn orLys); Xaa at res.60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val);Xaa at res.65=(Pro or Ala); Xaa at res.71=(Gln or Lys); Xaa atres.73=(Asn or Ser); Xaa at res.75 (Ile or Thr); Xaa at res.80=(Phe orTyr); Xaa at res.82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa atres.89=(Lys or Arg); Xaa at res.91=(Tyr or His); and Xaa at res.97=(Argor Lys).

[0043] In another embodiment, the morphogenic proteins comprise speciesof the generic amino acid sequence1        10        20         30        40         50CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX (SEQ ID NO:10)          60        70         80         90         100QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX

[0044] or residues 6-102 of SEQ ID NO:10, where the letters indicate theamino acid residues of standard single letter code, and the Xs representany amino acid residues. Cysteine residues are highlighted.

[0045] Preferred amino acid sequences within the foregoing genericsequence (SEQ ID NO:10) are:         10        20        30        40        50    LYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV       K S S L  QE VIS E FD Y  E A AY MPESMKAS   VI       F E K I  DN     L    N  S   Q  ITK F P    TL           A    S      K         60        70        80         90         100QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCGCR SI HAI SEQV EP    EQMNSLAI FFNDQDK I RK EE T DA H H    RF    T   S     K DPV V  Y N S     H RN   RS     N    S                       K       P     E and1        10        20        30        40        50CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV RRRS K S S L  QE VIS E FD Y  E A AY MPESMKAS   VI   KE F E K I  DN     L    N  S   Q  ITK F P    TLQ         A    S      K        60        70        80        90       100QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCGCR SI HAI SEQV EP    EQMNSLAI FFNDQDK I RK EE T DA H H    RF    T   S     K DPV V  Y N S     H RN   RS     N    S                      K       P     E

[0046] wherein each of the amino acids arranged vertically at eachposition in the sequence may be used alternatively in variouscombinations (SEQ ID NO:10). These generic sequences have 6 or 7cysteine residues where inter- or intra-molecular disulfide bonds canform. These sequences also contain other critical amino acids thatinfluence the tertiary structure of the proteins.

[0047] In still another embodiment, useful morphogenic proteins comprisean amino acid sequence encoded by a nucleic acid that hybridizes, underlow, medium or high stringency hybridization conditions, to DNA or RNAencoding reference morphogenic protein coding sequences. Exemplaryreference sequences include the C-terminal sequences defining theconserved seven-cysteine domains of OP-1, OP-2, BMP-2, BMP-4, BMP-5,BMP-6, 60A, GDF-3, GDF-5, GDF-6, GDF-7, and the like. High stringenthybridization conditions are herein defined as hybridization in 40%formamide, 5×SSPE, 5× Denhardt's Solution, and 0.1% SDS at 37° C.overnight, and washing in 0.1× SSPE, 0.1% SDS at 50° C. Standardstringency conditions are well characterized in commercially available,standard molecular cloning texts. See, for example, Molecular Cloning, ALaboratory Manual, 2nd Ed., ed. by Sambrook et al. (Cold Spring HarborLaboratory Press 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); and B. Perbal, APractical Guide To Molecular Cloning (1984).

[0048] Suitable in vitro, ex vivo and in vivo bioassays known in theart, including those described herein, may be used to ascertain whethera new BMP-related gene product has a morphogenic activity. Expressionand localization studies defining where and when the gene is expressedmay also be used to identify potential morphogenic activities. Nucleicacid and protein localization procedures are well known to those ofskill in the art (see, e.g., Ausubel et al., eds. Current Protocols inMolecular Cloning, Greene Publishing and Wiley Interscience, New York,1989).

[0049] Many of the identified BMPs are osteogenic and can induce boneand cartilage formation when implanted into mammals. Some BMPsidentified based on sequence homology to known osteogenic proteinspossess other morphogenic activities and a combination of a hormone anda soluble receptor thereof may be used to enhance those activities. Forexample, BMP-12 and BMP-13 reportedly induce ectopic formation oftendon/ligament-like tissue when implanted into mammals (Celeste et al.,WO 95/16035). Using this bioassay, a skilled practitioner can readilyidentify one or more combinations of hormones and soluble receptorsthereof that can stimulate the ability of the BMP to inducetendon/ligament-like tissue formation.

[0050] Certain BMPs which are known to be osteogenic can also induceneuronal cell differentiation. Embryonic mouse cells treated with BMP-2or OP-1 differentiate into astrocyte-like (glial) cells, and peripheralnerve regeneration using BMP-2 has been reported (Wang et al., WO95/05846). In addition, BMP-4, BMP-5 and OP-1 are expressed in epidermalectoderm flanking the neural plate. Ectopic recombinant BMP-4 and OP-1proteins can induce neural plate cells to initiate dorsal neural cellfate differentiation (Liem et al., Cell, 82, pp. 969-79 (1995)). At thespinal cord level, OP-1 and other BMPs can induce neural crest celldifferentiation. It is suggested that OP-1 and these BMPs can inducemany or all dorsal neural cell types, including roof plate cells, neuralcrest cells, and commissural neurons, depending on localized positionalcues.

[0051] That several osteogenic proteins originally derived from bonematrix are involved in neural development suggests that these and othermembers of the BMP family have additional tissue inductive propertiesthat are not yet disclosed. It is envisioned that the hormone/receptorcombinations set forth in this invention can be used to enhance new orknown tissue inductive properties of various known morphogenic proteins.It is also envisioned that the invention described herein will be usefulfor stimulating tissue inductive activities of new morphogenic proteinsas they are identified in the future.

[0052] Production of Morphogenic Proteins

[0053] The morphogenic proteins of this invention can be derived from avariety of sources. For instance, they may be isolated from naturalsources, recombinantly produced, or chemically synthesized.

[0054] A. Naturally Derived Morphogenic Proteins

[0055] The morphogenic proteins of the invention can be purified fromtissue sources, e.g., mammalian tissue sources, using well knowntechniques. See, e.g., Oppermann et al., U.S. Pat. Nos. 5,324,819 and5,354,557. If a purification protocol is unpublished, as for a newlyidentified morphogenic protein, conventional protein purificationtechniques (e.g., immunoaffinity) may be performed in combination withmorphogenic activity assays. Such assays allow the trace of themorphogenic activity through a series of purification steps.

[0056] B. Recombinantly Expressed Morphogenic Proteins

[0057] In another embodiment of this invention, the morphogenic proteinis produced by expressing an appropriate recombinant DNA molecule in ahost cell. The DNA and amino acid sequences of many BMPs and OPs havebeen reported, and methods for their recombinant production arepublished and otherwise known to those of skill in the art. For ageneral discussion of cloning and recombinant DNA technology, seeAusubel et al., supra; see also Watson et al., Recombinant DNA, 2d ed.1992 (W. H. Freeman and Co, New York).

[0058] The DNA sequences encoding bovine and human BMP-2 (formerlyBMP2A) and BMP-4 (formerly BMP-2B), and processes for recombinantlyproducing the corresponding proteins are described in U.S. Pat. Nos.5,011,691, 5,013,649, 5,166,058 and 5,168,050. The DNA and amino acidsequences of human and bovine BMP-5 and BMP-6, and methods for theirrecombinant production, are disclosed in U.S. Pat. No. 5,106,748, and5,187,076, respectively; see also U.S. Pat. Nos. 5,011,691 and5,344,654. Methods for OP-1 recombinant expression are disclosed inOppermann et al., U.S. Pat. Nos. 5,011,691 and 5,258,494. For analignment of BMP-2, BMP-4, BMP-5, BMP-6 and OP-1 (BMP-7) amino acidsequences, see WO 95/16034. DNA sequences encoding BMP-8 are disclosedin WO 91/18098, and DNA sequences encoding BMP-9 in WO 93/00432. DNA anddeduced amino acid sequences encoding BMP-10 and BMP-11 are disclosed inWO 94/26893, and WO 94/26892, respectively. DNA and deduced amino acidsequences for BMP-12 and BMP-13 are disclosed in WO 95/16035. The abovepatent disclosures, which describe DNA and amino acid sequences, andmethods for producing the BMPs and OPs encoded by those sequences, areincorporated herein by reference.

[0059] To clone genes that encode new BMPs, OPs and other morphogenicproteins identified in extracts by bioassay, methods entailing “reversegenetics” may be employed. Such methods start with a protein of known orunknown function to obtain the gene that encodes that protein. Standardprotein purification techniques may be used as an initial step. Ifenough protein can be purified to obtain a partial amino acid sequence,a degenerate DNA probe capable of hybridizing to the DNA sequence thatencodes that partial amino acid sequence may be designed, synthesizedand used as a probe to isolate full-length clones that encode that or arelated morphogenic protein.

[0060] Alternatively, a partially-purified extract containing themorphogenic protein may be used to raise antibodies directed againstthat protein. Morphogenic protein-specific antibodies may then be usedas a probe to screen expression libraries made from cDNAs (see, e.g.,Broome and Gilbert, Proc. Natl. Acad. Sci. U.S.A., 75, pp. 2746-49(1978); Young and Davis, Proc. Natl. Acad. Sci. U.S.A., 80, pp. 31-35(1983)).

[0061] For cloning and expressing new BMPs, OPs and other morphogenicproteins identified based on DNA sequence homology, the homologoussequences may be cloned and sequenced using standard recombinant DNAtechniques. With the DNA sequence available, a DNA fragment encoding themorphogenic protein may be inserted into an expression vector selectedto work in conjunction with a desired host expression system. The DNAfragment is cloned into the vector such that its transcription iscontrolled by a heterologous promoter in the vector, preferably apromoter which may be optionally regulated.

[0062] Some host-vector systems appropriate for the recombinantexpression of BMPs and OPs are disclosed in the references cited above.Useful host cells include but are not limited to bacteria such as E.coli, yeasts such as Saccharomyces and Picia, insects cells and otherprimary, transformed or immortalized eukaryotic cultured cells.Preferred eukaryotic host cells include CHO, COS and BSC cells (seebelow).

[0063] An appropriate vector is selected according to the host systemselected. Useful vectors include but are not limited to plasmids,cosmids, bacteriophage, insect and animal viral vectors, including thosederived from retroviruses and other single and double-stranded DNAviruses.

[0064] In one embodiment of this invention, the morphogenic protein maybe derived from a recombinant DNA molecule expressed in a prokaryotichost. Using recombinant DNA techniques, various fusion genes have beenconstructed to induce recombinant expression of naturally sourcedosteogenic sequences in E. coli (see, e.g., Oppermann et al., U.S. Pat.No. 5,354,557, incorporated herein by reference). Using analogousprocedures, DNAs comprising truncated forms of naturally sourcedmorphogenic sequences may be prepared as fusion constructs linked by asequence coding for the acid labile cleavage site (Asp-Pro) to a leadersequence (such as the “MLE leader”) suitable for promoting expression inE. coli.

[0065] In another embodiment of this invention, the morphogenic proteinis expressed using a mammalian host-vector system (e.g., transgenicproduction or tissue culture production). A morphogenic protein soexpressed may resemble more closely the naturally occurring protein.While the glycosylation pattern of the recombinant protein may sometimesdiffer from that of the natural protein, such differences are often notessential for biological activity of the recombinant protein. Techniquesfor transfection, expression and purification of recombinant proteinsare well known in the art. See, e.g., Ausubel et al., supra, and Bendig,Genetic Engineering, 7, pp. 91-127 (1988).

[0066] Mammalian DNA vectors should include appropriate sequences topromote expression of the gene of interest. Such sequences includetranscription initiation, termination and enhancer sequences; efficientRNA processing signals such as splicing and polyadenylation signals;mRNA-stabilizing sequences; translation-enhancing sequences (e.g., Kozakconsensus sequence); protein-stabilizing sequences; and when desired,sequences that enhance protein secretion.

[0067] Restriction maps and sources of various exemplary expressionvectors designed for OP-1 expression in mammalian cells have beendescribed in U.S. Pat. No. 5,354,557. Each of these vectors employs afull-length hOP-1 cDNA sequence inserted into the pUC-18 vector. It willbe appreciated by those of skill in the art that DNA sequences encodingtruncated forms of morphogenic proteins may also be used, provided thatthe expression vector or host cell provides the sequences necessary todirect processing and secretion of the expressed protein.

[0068] Useful promoters include, but are not limited to, the SV40 earlyand late promoters, the adenovirus-major late promoter, the mousemetallothionein-I (“mMT”) promoter, the Rous sarcoma virus (“RSV”) longterminal repeat (“LTR”), the mouse mammary tumor virus (“MMTV”) LTR, andthe human cytomegalovirus (“CMV”) major intermediate-early promoter. Forinstance, a combination of the CMV or MMTV promoter with an enhancersequence from the RSV LTR has been found to be particularly useful inexpressing human osteogenic proteins.

[0069] Preferred DNA vectors also include a marker gene (e.g., neomycinor DHFR) and means for amplifying the copy number of the gene ofinterest. DNA vectors may also comprise stabilizing sequences (e.g.,ori- or ARS-like sequences and telomere-like sequences), or mayalternatively be designed to favor directed or non-directed integrationinto the host cell genome.

[0070] One method of gene amplification in mammalian cell systems is theuse of the selectable dihydrofolate reductase (DHFR) gene in a dhfr⁻cell line. Generally, the DHFR gene is provided on the vector carryingthe gene of interest, and addition of increasing concentrations of thecytotoxic drug methotrexate (MTX) leads to amplification of the DHFRgene copy number, as well as that of the gene physically associated withit. DHFR as a selectable, amplifiable marker gene in transfected Chinesehamster ovary (CHO) cell lines is particularly well characterized in theart. Other useful amplifiable marker genes include the adenosinedeaminase (ADA) and glutamine synthetase (GS) genes.

[0071] Gene amplification can be further enhanced by modifying markergene expression regulatory sequences (e.g., enhancer, promoter, andtranscription or translation initiation sequences) to reduce the levelsof marker protein produced. Lowering the level of DHFR transcriptionincreases the DHFR gene copy number (and the physically-associated gene)to enable the transfected cell to adapt to growth in even low levels ofmethotrexate (e.g., 0.1 μM MTX). Preferred expression vectors such aspH754 and pH752 (Oppermann et al., U.S. Pat. No. 5,354,557, FIGS. 19Cand D) have been manipulated, using standard recombinant DNA technology,to create a weak DHFR promoter. As will be appreciated by those skilledin the art, other useful weak promoters, different from those disclosedherein, can be constructed using standard methods. Other regulatorysequences also can be modified to achieve the same effect.

[0072] Another gene amplification scheme relies on the temperaturesensitivity (ts) of BSC40-tsA58 cells transfected with an SV40 vector.Temperature reduction to 33° C. stabilizes the temperature-sensitiveSV40 T antigen, which leads to the excision and amplification of theintegrated transfected vector DNA, thereby amplifying thephysically-associated gene of interest.

[0073] The choice of cells/cell lines depends on the needs of theskilled practitioner. Monkey kidney cells (COS) provide high levels oftransient gene expression and are thus useful for rapidly testing vectorconstruction and the expression of cloned genes. COS cells expressingthe gene of interest can be established by transfecting the cells with,e.g., an SV40 vector carrying the gene. Stably transfected cell lines,on the other hand, can be used for long term production of morphogenicproteins. By way of example, both CHO cells and BSC40-tsA58 cells can beused as host cells. Recombinant OP-1 has been expressed in threedifferent cell expression systems: COS cells for rapidly screening thefunctionality of the various expression constructs, CHO cells for theestablishment of stable cell lines, and BSC40-tsA58 cells as analternative means of producing recombinant OP-1 protein.

[0074] Several bone-derived osteogenic proteins (OPs) and BMPs are foundas homo- and heterodimers comprising interchain disulfide bonds in theiractive forms. For instance, BMP-2, BMP-4, BMP-6 and BMP-7(OP-1)—originally isolated from bone—are bioactive as either homodimersor heterodimers. The ability of OPs and BMPs to form heterodimers mayconfer additional or altered morphogenic activities on morphogenicproteins. Heterodimers may exhibit qualitatively or quantitativelydifferent binding affinities than homodimers for OP and BMP receptors.Altered binding affinities may in turn result in differential activationof receptors that mediate different signalling pathways, ultimatelyleading to different biological activities. Altered binding affinitiescan also be manifested in a tissue or cell type-specific manner, therebyinducing only particular progenitor cell types to undergo proliferationand/or differentiation.

[0075] The dimeric proteins can be isolated from the culture mediaand/or refolded and dimerized in vitro to form biologically activecompositions. Heterodimers can be formed in vitro by combining separate,distinct polypeptide chains. Alternatively, heterodimers can be formedin a single cell by co-expressing nucleic acids encoding separate,distinct polypeptide chains. See, e.g., WO 93/09229 and U.S. Pat. No.5,411,941, for exemplary protocols for heterodimer protein production.

[0076] C. In Vivo Expression of Morphogenic Proteins

[0077] The morphogenic protein of the invention can also be produced invivo in a patient. To achieve this, an expression vector comprising apromoter operatively linked to a coding sequence of the morphogenicprotein may be introduced into progenitor cells in the patient.Alternatively, one can isolate the appropriate progenitor cells from thepatient, transfect or transduce the cells with the expression vector,and re-introduce the treated cells to the patient at a desired locus.

[0078] (1) Vectors

[0079] A nucleic acid construct according to this invention is derivedfrom a non-replicating linear or circular DNA or RNA vector, or from anautonomously replicating plasmid or viral vector. Alternatively, theconstruct is integrated into the host genome. Any vector that cantransfect or transduce the desired progenitor cell may be used.Preferred vectors are viral vectors, including those derived fromreplication-defective retroviruses (see, e.g., WO89/07136; Rosenberg etal., N. Eng. J. Med. 323(9): 570-578 (1990)), adenovirus (see, e.g.,Morsey et al., J. Cell. Biochem., Supp. 17E (1993)), adeno-associatedvirus (Kotin et al., Proc. Natl. Acad. Sci. USA 87:2211-2215 (1990)),replication-defective herpes simplex viruses (HSV; Lu et al., Abstract,page 66, Abstracts of the Meeting on Gene Therapy, Sep. 22-26, 1992,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), vaccinia virus(Mukherjee et al., Cancer Gene Ther. 7:663-70 (2000)), and any modifiedversions of these vectors. Methods for constructing expression vectorsare well known in the art. See, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 2ndEdition, Cold Spring Harbor, N.Y., 1989).

[0080] (2) Expression Control Sequences

[0081] In these vectors, expression control sequences are operablylinked to the nucleic acid sequence encoding the morphogenic proteinuseful in this invention. For eukaryotic cells, expression controlsequences may include a promoter, an enhancer, such as one derived froman immunoglobulin gene, SV40, cytomegalovirus, etc., and apolyadenylation sequence. A nucleic acid construct of this invention mayalso contain an internal ribosome entry site (“IRES”), and an intronthat may be desirably located between the promoter/enhancer sequence andthe morphogenic protein-coding sequence. Selection of these and othercommon vector elements are conventional. See, e.g., Sambrook et al,supra; Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, (1989); and references cited therein.

[0082] In one embodiment of the present invention, high-levelconstitutive expression is desired. Exemplary promoters for this purposeinclude, without limitation, the retroviral Rous sarcoma virus (RSV) LTRpromoter/enhancer, the cytomegalovirus (CMV) immediate earlypromoter/enhancer (see, e.g., Boshart et al, Cell 41:521-530 (1985)),the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmicβ-actin promoter, the phosphoglycerol kinase (PGK) promoter. Usefulpromoters for BMP expression in osteoblasts also include the Type Icollagen gene promoter and the CBFA gene promoter. Useful promoters forBMP expression in chondrocytes and chondroblasts include the Type IIcollagen gene promoter and the Type X collagen gene promoter. In anotherembodiment, the native transcription-regulatory elements of the desiredmorphogenic protein can be used.

[0083] Using the guidance provided by this application, one of skill inthe art may make a selection among the above expression controlsequences and modified versions thereof without departing from the scopeof this invention.

[0084] (3) Administration of Nucleic Acid Constructs

[0085] The nucleic acid constructs of this invention may be formulatedas a pharmaceutical composition for use in any form of transient and/orstable gene transfer in vivo and in vitro. The composition comprises atleast the nucleic acid construct and a pharmaceutically acceptablecarrier such as saline. Other aqueous and non-aqueous sterilesuspensions known to be pharmaceutically acceptable carriers and wellknown to those of skill in the art may be employed also. The constructmay be used for in vivo and ex vivo gene therapy, in vitro proteinproduction and diagnostic assays.

[0086] The nucleic acid construct can be introduced into target cells asnaked DNA, or by, e.g., liposome fusion (see, e.g., Nabel et al.,Science 249:1285-8 (1990); Ledley, J Pediatrics 110:1-8 and 167-74(1987); Nicolau et al., Proc Natl Acad Sci USA 80:1068-72 (1983)),erythrocyte ghosts, or microsphere methods. (microparticles; see, e.g.,U.S. Pat. No. 4,789,734; U.S. Pat. No. 4,925,673; U.S. Pat. No.3,625,214; Gregoriadis, Drug Carriers in Biology and Medicine, pp.287-341, Academic Press, 1979).

[0087] If the nucleic acid construct is viral-based, it can also bepackaged as a virion which then is used to transduce a cell (e.g., anautologous T cell isolated from a patient) in vitro. The infected cellis then introduced into the patient. Alternatively, the recombinantvirus may be administered to a patient directly, e.g., locally at thetissue defect site; or intravenously, intraperitoneally, intranasally,intramuscularly, subcutaneously, and/or intradermally, as determined byone skilled in the gene therapy art. A slow-release device, such as animplantable pump, may be used to facilitate delivery of the recombinantvirus to a cell. Where the virus is administered to a subject, thespecific cells to be infected may be targeted by controlling the methodof delivery. The treatments of the invention may be repeated as needed,as determined by one skilled in the art.

[0088] Dosages of the nucleic acid construct of this invention in genetherapy will depend primarily on factors such as the condition beingtreated. The dosage may also vary depending upon the age, weight andhealth of the patient. For example, an effective human dosage of aBMP-coding virus is generally in the range of from about 0.5 ml to 50 mlof saline solution containing the virus at concentrations of about1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, or1×10¹⁶ viral particles per dose administered. The dosage will beadjusted to balance the corrective benefits against any adverse sideeffects. The levels of expression of BMP may be monitored to determinethe type and frequency of dosage administration.

[0089] D. Synthetic Non-native Morphogenic Proteins

[0090] In another embodiment of this invention, a morphogenic proteinmay be prepared synthetically. Morphogenic proteins preparedsynthetically may be native, or may be non-native proteins, i.e., thosenot otherwise found in nature.

[0091] Non-native morphogenic proteins can be made by mutating nativemorphogenic proteins. Methods for making mutations that favor refoldingand/or assembling subunits into forms that exhibit greater morphogenicactivity have been described. See, e.g., U.S. Pat. No. 5,399,677.

[0092] Non-native morphogenic proteins can also be synthesized using aseries of consensus sequences (U.S. Pat. No. 5,324,819). These consensussequences were designed based on partial amino acid sequence dataobtained from native osteogenic products and on their homologies withother proteins reportedly having a presumed or demonstrateddevelopmental function. Several biosynthetic consensus sequences (calledconsensus osteogenic proteins or “COPs”) have been expressed as fusionproteins in prokaryotes. Purified fusion proteins may be cleaved,refolded, combined with a hormone and a soluble receptor thereof,implanted in an established animal model and examined for their bone-and/or cartilage-inducing activity. Certain preferred syntheticosteogenic proteins comprise one or both of two synthetic amino acidsequences designated COP5 (SEQ ID NO:11) and COP7 (SEQ ID NO: 12).

[0093] The amino acid sequences of COP5 and COP7 are shown below, as setforth in Oppermann et al., U.S. Pat. Nos. 5,011,691 and 5,324,819, whichare incorporated herein by reference: COP5LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD COP7LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD COP5HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA COP7HLNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA COP5 ISMLYLDENEKVVLKYNQEMVVEGCGCR(SEQ ID NO:11) COP7 ISMLYLDENEKVVLKYNQEMVVEGCGCR (SEQ ID NO:12)

[0094] In these amino acid sequences, the dashes (-) are used as fillersonly to line up comparable sequences in related proteins. Differencesbetween the aligned amino acid sequences are highlighted.

[0095] In one embodiment of this invention, the morphogenic protein is asynthetic osteogenic protein comprising a partial or complete sequenceof a generic sequence described above (SEQ ID NO:4, 5, 6, 7, or 10) suchthat it is capable of inducing tissue formation when properly folded andimplanted in a mammal. For instance, the synthetic protein can inducebone formation from osteoblasts when implanted in a favorableenvironment; or it can promote cartilage formation when implanted in anavascular locus or when co-administered with an inhibitor of full bonedevelopment.

[0096] In another embodiment, the synthetic morphogenic protein of thisinvention comprises a sequence sufficiently duplicative of a partial orcomplete sequence of a COP, e.g., COP5 (SEQ ID NO:11) or COP7 (SEQ IDNO:12). Biosynthetic COP sequences are believed to dimerize duringrefolding and appear not to be active when reduced. Both homodimeric andheterodimeric COPs are contemplated in this invention. In certainembodiments, this synthetic protein is less than about 200 amino acidslong.

[0097] These and other synthetic non-native osteogenic proteins may beused in concert with a hormone/receptor pair and tested using in vitro,ex vivo or in vivo bioassays for progenitor cell induction and tissueregeneration. The proteins in conjunction with the hormone/receptorpairs of this invention are envisioned to be useful for the repair andregeneration of bone, cartilage, tendon, ligament, neural andpotentially other types of tissue.

[0098] Homologous Proteins Having Morphogenic Activity

[0099] The morphogenic proteins useful in this invention may be producedby recombinant expression of DNA sequences isolated based on homologywith the osteogenic COP consensus sequences described above. SyntheticCOP DNA sequences may be used as probes to retrieve related DNAsequences from a variety of species (see, e.g., Oppermann et al., U.S.Pat. Nos. 5,011,591 and 5,258,494, which are incorporated herein byreference).

[0100] Morphogenic proteins encoded by a gene that hybridizes with a COPsequence probe are assembled into two subunits disulfide-bonded toproduce a heterodimer or homodimer capable of inducing tissue formationwhen implanted into a mammal. Recombinant BMP-2 and BMP-4 have beenshown to have cross-species osteogenic activity as homodimers and asheterodimers assembled with OP-1 subunits. Morphogenic protein-encodinggenes that hybridize to synthetic COP sequence probes include genesencoding Vg1, inhibin, DPP, OP-1, BMP-2 and BMP-4. Vg1 is a knownXenopus laevis morphogenic protein involved in early embryonicpatterning. Inhibin is another developmental gene that is a member ofthe BMP family of proteins from Xenopus laevis. DPP is an amino acidsequence encoded by a Drosophila gene responsible for development of thedorso-ventral pattern. OP-1, BMP-2 and BMP-4 are osteogenic proteinsthat can induce cartilage, bone and neural tissue formation.

[0101] In another embodiment of this invention, a morphogenic proteinmay comprise a polypeptide encoded by a nucleic acid that hybridizesunder stringent conditions to an “OPS” nucleic acid probe (Oppermann etal., U.S. Pat. No. 5,354,557). “OPS”—standing for OP-1 “short”—refers tothe portion of the human OP-1 protein defining the conserved 6 cysteineskeleton in the C-terminal active region (97 amino acids; SEQ ID NO:2,residues 335-431).

[0102] One example of a stringent hybridization condition ishybridization in 4× SSC at 65° C. (or 10° C. higher than the calculatedmelting temperature for a hybrid between the probe and a nucleic acidsequence containing no mis-matched base pairs), followed by washing in0.1× SSC at the hybridization temperature. Another stringenthybridization condition is hybridization in 50% formamide, 4× SSC at 42°C.

[0103] Thus, in view of this disclosure, the skilled practitioner canreadily design and synthesize genes, or isolate genes from cDNA orgenomic libraries that encode amino acid sequences having morphogenicactivity. These genes can be expressed in prokaryotic or eukaryotic hostcells to produce large quantities of active osteogenic or otherwisemorphogenic proteins. The recombinant proteins may be in native,truncated, mutant, fusion, or other active forms capable of inducingformation of bone, cartilage, or other types of tissue, as demonstratedby in vitro and ex vivo bioassays and in vivo implantation in mammals,including humans.

[0104] Hormones and Receptors Thereof

[0105] A hormone/receptor pair of this invention is capable ofstimulating the ability of a morphogenic protein to induce tissueformation from a progenitor cell. In a method of this invention, thetissue inductive activity of a morphogenic protein in a mammal isimproved by co-administering effective amounts of a hormone and asoluble receptor thereof. Alternatively, the morphogenic protein and thehormone/receptor pair are administered sequentially. It has been foundthat the synergism between a morphogenic protein and a hormone/receptorpair is preserved even if the morphogenic protein is administered 4 to 8hours before the hormone/receptor pair. The morphogenic protein, thehormone, and the hormone receptor can also be administered separately.

[0106] One or more hormone/receptor pairs can be selected for use inconcert with one or more morphogenic proteins according to the desiredtissue type to be induced and the site at which the treatment will beadministered. The particular choice of a morphogenicprotein(s)/hormone(s)/receptor(s) combination and the relativeconcentrations at which they are combined may be varied systematicallyto optimize the tissue type induced at a selected treatment site usingthe procedures described herein.

[0107] Hormones useful in this invention include, but are not limitedto, interleukins. 1 through 8, fibroblast growth factor, vascularendothelial growth factor, platelet-derived growth factor, TGF-β, andprostaglandins (e.g., E1 and E2). It may be preferred that the targetcell has a cell-surface receptor for the hormone. The hormones can alsobe morphogenic proteins such as GDFs; as a result, the composition ofthis invention will contain two morphogenic proteins and a solublereceptor of one of these proteins.

[0108] One preferred hormone/receptor pair of this invention isIL-6/sIL-6R. IL-6 is a member of a subfamily of multifunctionalhormones. It appears to play a role in both bone formation and boneresorption by affecting mitogenesis of target cells and regulating thesynthesis of other local factors. Clinical studies show that IL-6 isinvolved in a variety of diseases, such as fibrous dysplasia,osteopenia, osteoporosis and Paget's disease. Recombinant full lengthhuman IL-6 (26 kD) expressed from E. coli can be obtained from Sigma(St. Louis, Mo.) and Promega (Madison, Wis.). Recombinant sIL-6Rproduced from baculovirus and containing the entire extracellular domain(residues 1-339; 38 kD) of human IL-6R can be obtained from Sigma andR&D Systems (Minneapolis, Minn.). See also Examples 1 and 2, infra.Active allelic, species or other variants of these IL-6 and sIL-6products can also be used.

[0109] The hormone or the hormone receptor of this invention can beassociated with an agent that is capable of increasing the hormone's orreceptor's bio-activity, e.g., synthesis, half-life, bio-availability,and reactivity with other bio-molecules such as binding proteins andreceptors. These agents may contain carrier molecules such as proteinsand lipids.

[0110] The hormone and hormone receptor are present in amounts capableof synergistically stimulating the tissue inductive activity of themorphogenic protein in a mammal. The relative concentrations ofmorphogenic protein, hormone and hormone receptor that optimally inducetissue formation may be determined empirically by the skilledpractitioner using the procedures described herein.

[0111] Progenitor Cells

[0112] The progenitor cell that is induced to proliferate and/ordifferentiate by the morphogenic protein of this invention is preferablya mammalian cell. Examples of useful progenitor cells are mammalianchondroblasts, osteoblasts and neuroblasts, all earlier developmentalprecursors thereof, and all cells that develop therefrom (e.g.,prechondroblasts and chondrocytes). The progenitor cell may be inducedto form one or more tissue types such as endochondral or intramembranousbone, cartilage, tendon/ligament-like tissue, neural tissue and kidneytissue. The specific morphogenic activity exhibited by a morphogenicprotein will depend in part on the type of the progenitor cell as wellas the treatment site. These variables may be tested empirically.

[0113] Morphogenic proteins are highly conserved throughout evolution,and non-mammalian progenitor cells are likely to be stimulated by same-or cross-species morphogenic proteins and hormone/receptor combinations.It is thus envisioned that where schemes are available for implantingxenogeneic cells into humans without adverse immunological reactions,non-mammalian progenitor cells stimulated by morphogenic protein and ahormone/receptor pair according to the procedures set forth herein willbe useful for tissue regeneration and repair in humans.

[0114] Testing Morphogenic Activity

[0115] To identify a hormone/receptor pair capable of stimulating thetissue inductive activity of a chosen morphogenic protein, anappropriate assay is selected. Initially, in vitro assays can beperformed. A useful in vitro assay may monitor a nucleic acid or proteinmarker whose expression is known to correlate with the associated celldifferentiation pathway. See, e.g., Examples 3 and 4 of U.S. Pat. No.5,854,207, Lee et al.; and Examples 1 and 2, infra.

[0116] Examples 1 and 2, infra, describe experiments using OP-1 toidentify and to optimize an effective concentration of IL-6 and sIL-6R.OP-1 is known to have osteogenic and neurogenic activity. Thus, toidentify a hormone/receptor pair having synergistic effects with OP-1,one can conduct an in vitro assay that examines the expression of amolecular marker, e.g., an osteogenic- or a neurogenic-associatedmarker, in appropriate progenitor cells.

[0117] One useful assay for testing potential hormone/receptor pairswith OP-1 for osteogenic activity is the alkaline phosphatase (“AP”)enzymatic assay. AP is an osteoblast differentiation marker in primaryosteoblastic fetal rat calvarial (“FRC”) cells. The OP-1stimulated APactivity results from increased steady-state AP mRNA levels. Otheruseful protein markers for monitoring osteogenic activity of acomposition include, but are not limited to, type I collagen,osteocalcin, osteopontin, bone sialoprotein and PTH-dependent cAMPlevels.

[0118] An AP assay is performed generally as follows. First, ahormone/receptor pair is identified by picking various concentrationsand ratios of the hormone and hormone receptor and testing them in theabsence and presence of a morphogenic protein. Second, the amounts ofhormone and hormone receptor required to achieve optimal, preferablysynergistic, tissue induction in concert with the morphogenic protein isdetermined by generating dose response curves.

[0119] Optionally, additional hormone/receptor pairs that furtherstimulate or otherwise alter the morphogenic activity induced by amorphogenic protein and a first hormone/receptor pair may be identifiedand a new multi-factor dose response curve generated. See, e.g., Example5 of U.S. Pat. No. 5,854,207.

[0120] Bone Induction Assays

[0121] The various morphogenic compositions and devices of thisinvention can also be evaluated with ex vivo or in vivo bioassays. A ratbioassay for bone induction may be used to monitor osteogenic activityof osteogenic proteins in concert with one or more hormone/receptorpairs. See, e.g., Sampath et al., Proc. Natl. Acad. Sci. USA, 80, pp.6591-95 (1983), and U.S. Pat. No. 5,854,207, Example 7. Rat bioassaysare useful as the first step in moving from in vitro studies to in vivostudies.

[0122] Large animal efficacy models for osteogenic device testing areknown in the art. Exemplary models are the feline femoral model, therabbit ulnar model, the dog ulnar model and the monkey model. See, e.g.,U.S. Pat. No. 5,354,557, and 5,854,207 (Examples 10 and 11 therein).

[0123] In general, about 500-1000 ng of active morphogenic protein andabout 10-200 ng of active hormone and active hormone receptor arecombined with 25 mg of a carrier matrix for rat bioassays. In largeranimals, typically about 0.8-Img of active morphogenic protein per gramof carrier is combined with about 100 ng or more of an active hormoneand hormone receptor. The optimal ratios of morphogenic protein tohormone and of hormone to hormone receptor for a specific tissue typemay be determined empirically by those of skill in the art according tothe procedures set forth herein. Greater amounts may be used for largeimplants.

[0124] Tendon/Ligament-Like Tissue Formation Bioassay

[0125] Assays for monitoring tendon and ligament-like tissue formationinduced by morphogenic proteins are known in the art. See, e.g., Celesteet al., WO 95/16035, hereby incorporated by reference. Such assays canbe used to identify hormone/receptor pairs that stimulatetendon/ligament-like tissue formation by BMP-12, BMP-13 or othermorphogenic proteins in a particular treatment site. The assays may alsobe used to optimize concentrations and treatment schedules fortherapeutic tissue repair regiments.

[0126] These assays may be used to test various combinations ofmorphogenic protein and hormone/receptor combinations, and to produce anin vivo dose response curve for determining the effective relativeconcentrations of morphogenic proteins and hormones/receptors.

[0127] Neural Assays

[0128] The osteogenic proteins BMP-4 and BMP-7 (OP-1) can induce ventralneural plate explants to undergo differentiation into dorsal neural cellfates (Liem et al., Cell, 82, pp. 969-79 (1995)). Molecular markers ofdorsal cell differentiation are described in Liem et al. These markersinclude PAX3 and MSX, whose expression delineates an early stage ofneural plate cell differentiation; DSL-1, a BMP-like moleculedelineating differentiation of dorsal neural plate cells at a stageafter neural tube closure; and SLUG protein, whose expression afterneural tube closure defines pre-migratory neural crest cells. Expressionof these dorsal markers can be induced in ventral neural plate explantsby ectopic BMP4 and OP-1.

[0129] A peripheral nerve regeneration assay using BMP-2 has beendescribed (Wang et al., WO 95/05846, hereby incorporated by reference).The assay involves the implantation of neurogenic devices in thevicinity of severed sciatic nerves in rats. This procedure may be usedto assess the ability of a putative hormone/receptor pair to enhance theneuronal inductive activity of homo- and heterodimers of morphogenicproteins having neurogenic activity, such as BMP-2, BMP-4, BMP-6 andOP-1, or of any selected neurogenic protein/hormone/hormone receptorcombinations.

[0130] Pharmaceutical Compositions

[0131] The pharmaceutical compositions of this invention contain atleast one (e.g., at least 2, 3 or 5) morphogenic protein, and at leastone (e.g., at least 2 or 3) hormone/receptor pair. These compositionsare capable of inducing tissue formation when administered, e.g.,implanted, into a patient. The compositions will be administered at aneffective dose to induce the particular type of tissue at the desiredtreatment site. Determination of a preferred pharmaceutical formulationand a therapeutically efficient dose regiment for a given application iswell within the skill of the art. Factors that need to be consideredinclude, for example, the administration mode, the condition and weightof the patient, the extent of desired treatment and the tolerance of thepatient for the treatment.

[0132] Doses expected to be suitable starting points for optimizingtreatment regiments are based on the results of in vitro assays, and exvivo or in vivo assays. Based on the results of such assays, a range ofsuitable morphogenic protein and hormone/receptor concentration ratioscan be selected to test at a treatment site in animals and then inhumans.

[0133] The pharmaceutical compositions of this invention may be in avariety of forms. These include, for example, solid, semi-solid andliquid forms such as tablets, pills, powders, liquids, suspensions,suppositories, gels, pastes, and other injectable and infusiblesolutions. The preferred form depends on the intended mode ofadministration and therapeutic application. Modes of administration mayinclude oral, parenteral, subcutaneous, intravenous, intralesional ortopical administration. In most cases, the pharmaceutical compositionsof this invention will be administered in the vicinity of or at thetreatment site in need of tissue regeneration or repair.

[0134] The pharmaceutical compositions of this invention may, forexample, be placed into sterile, isotonic formulations with or withoutco-factors which stimulate uptake or stability. For example, thecompositions may contain a formulation buffer comprising 5.0 mg/mlcitric acid monohydrate, 2.7 mg/ml trisodium citrate, 41 mg/ml mannitol,1 mg/ml glycine and 1 mg/ml polysorbate 20. This solution can belyophilized, stored under refrigeration and reconstituted prior toadministration with sterile Water-For-Injection (USP).

[0135] The compositions may also include pharmaceutically acceptablecarriers well known in the art. See, for example, Remington'sPharmaceutical Sciences, 16th Edition, 1980, Mac Publishing Company.Such pharmaceutically acceptable carriers may include other medicinalagents, carriers, genetic carriers, adjuvants, and excipients such ashuman serum albumin or plasma preparations. The compositions may be inthe form of a unit dose and will usually be administered as a doseregiment that depends on the particular tissue treatment.

[0136] The pharmaceutical compositions of this invention may also beadministered in form of a morphogenic device using, for example,microspheres, liposomes, other microparticulate delivery systems orsustained release formulations placed in, near, or otherwise incommunication with affected tissues or the bloodstream bathing thosetissues.

[0137] Liposomes containing the polypeptide mixtures of this inventioncan be prepared by well-known methods. See, e.g. DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. U.S.A., 82, pp. 3688-92 (1985); Hwang etal., Proc. Natl. Acad. Sci. U.S.A., 77, pp. 4030-34 (1980); U.S. Pat.Nos. 4,485,045 and 4,544,545. Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol. The proportion of cholesterolis selected to control the optimal release rate of the polypeptides ofinterest.

[0138] The polypeptide mixtures of this invention may also be attachedto liposomes containing other biologically active molecules to modulatethe rate and characteristics of tissue induction. Such attachment may beaccomplished by cross-linking agents such as heterobifunctionalcross-linking agents that have been widely used to couple toxins orchemotherapeutic agents to antibodies for targeted delivery. Conjugationto liposomes can also be accomplished using the carbohydrate-directedcross-linking reagent 4-(4-maleimidophenyl)butyric acid hydrazide. See,e.g., Duzgunes et al., J. Cell. Biochem. Abst., Suppl. 16E 77 (1992).

[0139] Morphogenic Devices

[0140] The pharmaceutical compositions of this invention canadditionally contain an implantable, biocompatible carrier. Suchcompositions are also called morphogenic devices. The carrier functionsas a sustained release delivery system for the therapeutic proteins andprotects the proteins from non-specific proteolysis. The carrier may bebiodegradable in vivo. A sustained release carrier may containsemipermeable polymer matrices in the form of shaped articles, e.g.,suppositories or capsules. Such a carrier can be made of polylactides(U.S. Pat. No. 3,773,319; EP 58,481), copolymers of L-glutamic acid andethyl-L-glutamate (Sidman et al., Biopolymers, 22, pp. 547-56 (1985));poly(2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer etal., J. Biomed. Mater. Res., 15, pp. 167-277 (1981); Langer, Chem.Tech., 12, pp. 98-105 (1982)).

[0141] The carrier may also serve as a temporary scaffold and substratumfor recruitment of migratory progenitor cells and their subsequentanchoring and proliferation, until replaced by new bone or otherappropriate tissue. For example, the carrier may contain a biocompatiblematrix made up of particles or otherwise having the desired porosity ormicrotexture. The pores will permit migration, anchoring,differentiation and proliferation of the relevant progenitor cells. Theparticle size may be within the range of 70 μm-850 μm, e.g., 70 μm-420μm or 150 μm-420 μm. A particulate matrix may be fabricated by closepacking particulate materials into a shape spanning the tissue defect tobe treated. Various matrices known in the art can be employed. See,e.g., U.S. Pat. Nos. 4,975,526, 5,162,114 and 5,171,574, and WO91/18558, all of which are herein incorporated by reference.

[0142] Useful matrix materials include but are not limited to collagen;celluloses, including carboxymethyl cellulose; homo- or co-polymers ofglycolic acid, lactic acid, and butyric acid, including derivativesthereof; and ceramics, such as hydroxyapatite, tricalcium phosphate andother calcium phosphates. See, e.g., U.S. Pat. No. 5,854,207, col. 25,line 59, through col. 27, line 6. Various combinations of these or othersuitable matrix materials may be useful as determined by the assays setforth herein. The choice of material depends in part on its in vivodissolution rate. In bones, the dissolution rates can vary according towhether the implant is placed in cortical or trabecular bone.

[0143] Other useful matrices include particulate, demineralized,guanidine-extracted, allogenic bone; and specially treated, particulate,protein-extracted, demineralized xenogenic bone. See, e.g., Example 6 ofU.S. Pat. No. 5,854,207. Such xenogenic bone powder matrices may betreated with proteases such as trypsin. Preferably, the xenogenicmatrices are treated with one or more fibril-modifying agents toincrease the intraparticle intrusion volume (porosity) and surface area.Useful modifying agents include solvents such as dichloromethane,trichloroacetic acid, acetonitrile and acids such as trifluoroaceticacid and hydrogen fluoride. A preferred fibril-modifying agent is in theform of a heated aqueous medium, preferably an acidic aqueous mediumhaving a pH less than about 4.5, most preferably having a pH betweenabout 2 and 4, inclusive. The acidic aqueous medium can, for instance,be 0.1% acetic acid with a pH of about 3. Heating demineralized,delipidated, guanidine-extracted bone collagen in an aqueous medium atelevated temperatures (e.g., at about 37° C.-65° C., preferably at about45° C.-60° C.) for approximately one hour is generally sufficient toachieve the desired surface morphology. It is hypothesized that the heattreatment alters the collagen fibrils, resulting in an increase in theparticle surface area.

[0144] Xenogenic bone matrices can be used in a variety of clinicalsettings. In addition to its use as a matrix for bone formation invarious orthopedic, periodontal, and reconstructive procedures, thematrix also may be used as a sustained release carrier, or as acollagenous coating for orthopedic or general prosthetic implants.

[0145] Demineralized guanidine-extracted xenogenic bovine bone containsa mixture of additional materials that may be fractionated further usingstandard biomolecular purification techniques. For example, boneextracts can be fractionated by chromatography, and the various extractfractions corresponding to the chromatogram peaks can be added backtogether to an active matrix. Doing so may remove inhibitors of bone ortissue-inductive activity, thereby improving matrix properties.

[0146] Besides morphogenic proteins and hormone/receptor pairs, themorphogenic devices of this invention may additionally contain otherhormones and trophic agents. The devices may also contain antibiotics,chemotherapeutic agents, enzymes, enzyme inhibitors and other bioactiveagents. These ingredients may be adsorbed onto or dispersed within thecarrier, and will be released over time at the implantation site as thecarrier material is slowly absorbed.

[0147] General Consideration of Matrix Properties

[0148] Factors influencing the performance of a matrix include matrixgeometry, particle size (if the matrix is made up of particles), themethodology for combining the matrix and morphogenic proteins, thedegree of both intra- and inter-particle porosity, the presence ofmineral, and the presence of surface charge. For example, studies haveshown that, in bone induction using OP-1 and a morphogenic proteinstimulating factor, perturbation of the matrix charge by chemicalmodifications can abolish bone inductive responses. Particle size alsoinfluences the quantitative response of new bone, with sizes between 701μm and 420 μm capable of eliciting the maximum response. Further,contamination of the matrix with bone mineral may inhibit boneformation. Individual heavy metal concentrations in a bone matrix can bereduced to less than about 1 ppm by the methods described herein.

[0149] The sequential cellular reactions at the interface of the bonematrix and an osteogenic protein implant are complex. The multi-stepcascade includes: binding of fibrin and fibronectin to the implantedmatrix, migration and proliferation of mesenchymal cells,differentiation of the progenitor cells into chondroblasts, cartilageformation, cartilage calcification, vascular invasion, bone formation,remodeling, and bone marrow differentiation. A successful matrix iscapable of accommodating each of these steps.

[0150] The matrix may be shaped as desired in anticipation of surgery orshaped by the physician or technician during surgery. It has been shownthat new bone is formed essentially with the dimensions of the implanteddevice. In the case where the matrix material is biodegradable in vivo,the matrix material is slowly absorbed by the body and is replaced bynew bone in the shape of, or very nearly the shape of, the implant.Thus, the matrix is preferably shaped to span a tissue defect and totake the desired form of the new tissue. For example, in the case ofbone repair of a non-union defect, it is desirable to use dimensionsthat span the non-union, and the new bone will eventually fill thedefect.

[0151] The matrix may be a shape-retaining solid made of loosely-adheredparticulate material, e.g., collagen. Alternatively, the matrix may be amolded, porous solid, or an aggregation of close-packed particles heldin place by surrounding tissue. Masticated muscle or other tissue mayalso be used. Large allogenic bone implants can act as a carrier for thematrix if their marrow cavities are cleaned and packed with particlescontaining dispersed osteogenic protein and hormone/receptor pair.

[0152] The matrix may also take the form of a paste or a hydrogel.“Hydrogel” refers to a three dimensional network of cross-linkedhydrophilic polymers in the form of a gel. The gel is substantiallycomposed of water, for instance, greater than 90% water. Hydrogelmatrices can carry a net positive or net negative charge, or may beneutral. A typical net negative charged matrix is alginate. Hydrogelscarrying a net positive charge are, for example, extracellular matrixcomponents such as collagen and laminin. Examples of commerciallyavailable extracellular matrix components include MATRIGEL™ andVITROGEN™. Example of a net neutral hydrogel are highly cross-linkedpolyethylene oxide and polyvinyl alcohol.

[0153] Prosthetic Devices

[0154] This invention also features an implantable prosthetic devicecomprising at least one morphogenic protein and at least onehormone/receptor pair at therapeutic amounts and ratios. The device canbe used in conjunction with a composition containing the same or othermorphogenic protein or hormone/receptor pair. The prosthetic device maybe made from a material containing metal or ceramic. Exemplaryprosthetic devices are hip devices, screws, rods and titanium cages forspine fusion. The device is implanted in a mammal (e.g., a human) at alocus where the target tissue and the surface of the prosthetic deviceare maintained at least partially in contact for a time sufficient topermit enhanced tissue growth between the target tissue and the device.

[0155] The osteogenic composition may be disposed on the prostheticimplant on a surface region that is to be positioned next to a targettissue in the mammal. Preferably, the mammal is a human patient. Thecomposition is disposed in an amount sufficient to promote enhancedtissue growth into the implant or onto its surface. The amount of thecomposition to be used may be determined empirically by using bioassayssuch as those described herein and in Rueger et al., U.S. Pat. No.5,344,654, which is incorporated herein by reference. Preferably, animalstudies are performed to optimize the concentration of the ingredientsin the device before a similar prosthetic device is used in a humanpatient. The prosthetic devices will be useful for repairing orthopedicdefects, injuries or anomalies in the treated mammal.

[0156] Utility of Morphogenic Compositions and Devices

[0157] The compositions, devices and methods of this invention willpermit a physician to treat a variety of tissue injuries, tissuedegenerations, and other diseased tissue conditions. The compositionsand devices can ameliorate or remedy these conditions by stimulatinglocal tissue formation or regeneration.

[0158] The devices of this invention may be implanted at the desiredlocus in a mammal such that the implant is accessible to the appropriateprogenitor cells of this mammal. The devices may be used alone or incombination with other therapies for tissue repair and regeneration.

[0159] The morphogenic devices of this invention may also be implantedin or surrounding a joint for use in cartilage and soft tissue repair,or in or surrounding nervous system-associated tissue for use in neuralregeneration and repair. The tissue specificity of the particularmorphogenic protein—or combination of morphogenic proteins with otherbiological factors—will determine the cell types or tissues that will beamenable to such treatments and can be selected by one skilled in theart. The ability to enhance morphogenic protein-induced tissueregeneration by co-administering a hormone/receptor pair according tothe present invention is thus not believed to be limited to anyparticular cell-type or tissue.

[0160] The osteogenic compositions and devices of this invention willpermit the physician to obtain predictable bone, ligament and/orcartilage formation using less osteogenic protein to achieve at leastabout the same extent of bone or cartilage formation. The osteogeniccompositions and devices of this invention may be used to treat moreeffectively the injuries, anomalies and disorders that have beendescribed in the prior art of osteogenic devices. These include, forexample, forming local bone in fractures, non-union fractures, fusionsand bony voids such as those created in tumor resections or thoseresulting from cysts; treating acquired and congenital craniofacial andother skeletal or dental anomalies (see e.g., Glowacki et al., Lancet,1, pp. 959-63 (1981)); performing dental and periodontal reconstructionswhere lost bone replacement or bone augmentation is required such as ina jaw bone; and supplementing alveolar bone loss resulting fromperiodontal disease to delay or prevent tooth loss (see e.g., Sigurdssonet al., J Periodontol., 66, pp. 511-21 (1995)).

[0161] An osteogenic device of this invention that comprises a matrixcomprising allogenic bone may also be implanted at a site in need ofbone replacement to accelerate allograft repair and incorporation in amammal. Another potential clinical application of the improvedosteogenic devices of this invention is in cartilage repair, forexample, following joint injury or in the treatment of osteoarthritis.The ability to enhance the cartilage-inducing activity of morphogenicproteins by co-administering a hormone/receptor pair may permit fasteror more extensive tissue repair and replacement using the same or lowerlevels of morphogenic proteins.

[0162] The morphogenic compositions and devices of this invention willbe useful in treating certain congenital diseases and developmentalabnormalities of cartilage, bone and other tissues. For example,homozygous OP-1-deficient mice die within 24 hours after birth due tokidney failure (Luo et al., J Bone Min. Res., 10 (Supp. 1), pp. S163(1995)). Kidney failure in these mice is associated with the failure toform renal glomeruli due to lack of mesenchymal tissue condensation.OP-1-deficient mice also have various skeletal abnormalities associatedwith their hindlimbs, rib cage and skull, are polydactyl, and exhibitaberrant retinal development. These results, in combination with thosediscussed above concerning the ability of OP-1 to induce differentiationinto dorsal neural cell fates, indicate that OP-1 plays an importantrole in epithelial-mesenchymal interactions during development. It isanticipated that the compositions, devices and methods of this inventionwill be useful for ameliorating these and other developmentalabnormalities.

[0163] Developmental abnormalities of the bone may affect isolated ormultiple regions of the skeleton or of a particular supportive orconnective tissue type. These abnormalities often require complicatedbone transplantation procedures and orthopedic devices. The tissuerepair and regeneration required after such procedures may occur morequickly and completely with the use of morphogenic compositions, devicesand methods of this invention.

[0164] Examples of heritable conditions, including congenital bonediseases, for which use of the morphogenic compositions and devices ofthis invention will be useful include osteogenesis imperfecta, theHurler and Marfan syndromes, and several disorders of epiphyseal andmetaphyseal growth centers such as is presented in hypophosphatasia, adeficiency in alkaline phosphatase enzymatic activity.

[0165] Inflammatory joint diseases may also benefit from the improvedmethods, compositions and devices of this invention. These diseasesinclude but are not limited to rheumatoid and psoriatic arthritis,bursitis, ulcerative colitis, regional enteritis, Whipple's disease,ankylosing spondylitis (also called Marie Strümpell or Bechterew'sdisease), and the so-called “collagen diseases” such as systemic lupuserythematosus (SLE), progressive systemic sclerosis (scleroderma),polymyositis (dermatomyositis), necrotizing vasculitides, Sjögren'ssyndrome (sicca syndrome), rheumatic fever, amyloidosis, thromboticthrombocytopenic purpura and relapsing polychondritis. Heritabledisorders of connective tissue include Marfan's syndrome,homocystinuria, Ehlers-Danlos syndrome, osteogenesis imperfecta,alkaptonuria, pseudoxanthoma elasticum, cutis laxa, Hurler's syndrome,and myositis ossificans progressiva.

EXAMPLES

[0166] The following are examples which illustrate the morphogeniccompositions and devices of this invention, and methods used tocharacterize them. These examples should not be construed as limiting.They are included for purposes of illustration and the present inventionis limited only by the claims.

Example 1

[0167]FIG. 1 shows the effects of IL-6, sIL-6R, and mixtures ofrecombinant human IL-6 and recombinant human sIL-6R (“IL-6/R”),respectively, on the OP-1-induced AP activity in FRC cells. ConfluentFRC cells were treated with the indicated agent(s) for 24 hrs. Theconcentrations of agent(s) used (ng/ml) are indicated in parentheses.For IL-6/R, the molar ratio of the two was maintained at about 1.2, andtheir respective amounts are indicated also in parentheses. Total APactivity was determined spectrophotometrically. Total cellular proteinwas determined by the Bradford Assay. Specific AP activity wascalculated as AP/protein unit. AP activity values were normalized tothat of 150 ng/ml OP-1 (=1) and represent the means of 8-12 independentdeterminations using 3 different FRC cell preparations.

[0168] As shown in FIG. 1, IL-6 alone in the concentration range testeddid not stimulate the basal AP activity. SIL-6R alone stimulated thebasal AP activity slightly; however, the stimulation did not seem to besIL-6R dose-dependent.

[0169]FIG. 1 further demonstrates that IL-6 potentiates the OP-1-inducedAP activity in a dose-dependent manner. A maximum of about 2-foldstimulation was observed (p<0.05). SIL-6R also potentiated theOP-1-induced AP activity in a dose-dependent manner. A higher fold(about 3.5-fold; p<0.02) of stimulation than observed with IL-6 wasachieved.

[0170] The effect of IL-6/R on the OP-1-induced AP activity was alsoexamined. At the highest tested dose of IL-6 plus its soluble receptor(10 ng/ml IL-6 and 125 ng/ml sIL-6R), the OP-1-induced AP activity wassynergistically enhanced by about 10-fold. This enhancement wasreproducible. However, at the lower dose range, the IL-6/R combinationdid not appear to stimulate beyond what was achieved by either IL-6 orits receptor alone; on the contrary, the combination appeared tosuppress AP activity.

Example 2

[0171]FIG. 2 shows that IL-6 alone enhanced OP-1 action in a mineralizedbone nodule formation assay. FRC cells were grown in αMEN (supplementedwith 5% FBS, 30 μg/ml gentamycin, 100 μg/ml ascorbic acid and 5 mMβ-glycerolphosphate), and treated for various durations of time with (1)solvent vehicle, (2) 200 ng/ml OP-1, or (3) 200 ng/ml OP-1 plus IL-6 atvarious concentrations. The culture media were replenished with the sametreatment agent(s) every three days. Progress of nodule formation wasmonitored every three days. After a total of 15 days, cells were fixedwith formalin and photographed. As seen in FIG. 3, IL-6/R also enhancedOP-1's ability to induce the formation of mineralized bone nodules.

Example 3

[0172] To determine whether IL-6/R effects its synergy with OP-1 bydirectly stimulating OP-1 responsive cells or by increasing the numberof OP-1 responsive cells, primary cultures of FRC were used as a modelsystem, in which AP activity levels were used as a biochemical marker ofOP-1 responsiveness. Histochemical data showed that the number of APpositive cells in cultures treated with IL-6/R (40 ng/ml IL-6 and 50ng/ml sIL-6R) and OP-1 (200 ng/ml) was similar to that in culturestreated with OP-1 alone (200 ng/ml). However, the AP activity level washigher in the former cultures than the latter cultures. IL-6 alone (40ng/ml) did not stimulate AP positive cells; and sIL-6R alone (50 ng/ml)or IL-6R (40 ng/ml IL-6 and 50 ng/ml sIL-6R) stimulated AP positivecells to a smaller extent, as compared to the combination of IL-6R andOP-1. These data suggest that IL-6/R's synergistic effect on OP-1results from IL-6/R's direct stimulation of OP-1 responsive cells.

Example 4

[0173] To investigate whether IL-6/R stimulates the expression of OP-1receptors on FRC cells, the mRNA levels of three BMP type I receptors(BMPR-IA, BMPR-IB, and ActR-I) and one BMP type II receptor (BMPR-II)were measured by Northern blot analysis.

[0174] Briefly, confluent FRC cells were treated for 48 hours with (1) avehicle; (2) 200 ng/ml OP-1; (3) 40 ng/ml IL-6 and 50 ng/ml sIL-6R; or(4) 200 ng/ml OP-1, 40 ng/ml IL-6, and 50 ng/ml sIL-6R. Total RNA wasisolated using the TR1 reagent (Sigma) and loaded onto an AGAROSE GTG(FMC) gel containing formaldehyde. Northern blots were prepared andprobed with ³²P-labeled cDNA encoding for the various BMPRs. Theseprobes hybridized only to mRNA. The radioactive bands were detected andquantified using a PHOSPHORIMAGER (Molecular Dynamics, Sunnyvale,Calif.). To normalize the band intensity of the BMPR bands, the blotswere also probed with an oligonucleotide for the 18S rRNA.

[0175] The mRNA levels in control FRC cells, OP-1-treated cells,IL-6/R-treated cells, and (OP-1+IL-6/R)-treated cells were compared(FIG. 4). The data showed that OP-1 did not affect the mRNA level of thetype I receptors, but stimulated the BMPR-II mRNA level by about 2.2fold. Likewise, IL-6/R did not alter the mRNA expression level of thetype I receptors, but increased the BMPR-II mRNA level by about1.5-fold. In the presence of OP-1 and IL-6/R, the mRNA level of the typeI receptors was not significantly changed; however, the BMPR-II mRNAlevel was almost 3-fold higher than the control. These results suggestthat IL-6/R can stimulate the osteogenic activity of OP-1 by elevatingBMPR-II mRNA expression.

Example 5

[0176] The OP-1 protein used in Examples 1-5 was provided exogenously tothe test cells. To investigate whether the same IL-6/R synergisticeffect would be observed when the OP-1 protein was expressedintracellularly in test cells, FRC cells were transfected with pW24, aplasmid carrying an OP-1 coding sequence under the control of the CMVpromoter.

[0177] Briefly, confluent FRC cells were transfected with pW24 (2μg/ml). After recovery, the transfected cells were treated withexogenous sIL-6R alone or IL-6/R for 24 hours. Then the total APactivity levels were determined (FIG. 5).

[0178] The data showed that the levels of OP-1-induced AP activity inpW24transfected cells were enhanced by sIL-6R in a dose-dependentmanner. At a concentration of 75 ng/ml, sIL-6R stimulated theOP-1-induced AP activity by as much as 4 fold.

[0179] The data also showed that the levels of OP-1-induced AP activityin pW24transfected cells were also enhanced by IL-6/R in adose-dependent manner. A 2.5-fold stimulation of the OP-1-induced APactivity was observed when IL-6/R was applied to the test cells atconcentrations of 60 ng/ml for IL-6 and 75 ng/ml for sIL-6R.

1 14 1 1822 DNA Homo sapiens CDS (49)..(1341) 1 ggtgcgggcc cggagcccggagcccgggta gcgcgtagag ccggcgcg atg cac gtg 57 Met His Val 1 cgc tca ctgcga gct gcg gcg ccg cac agc ttc gtg gcg ctc tgg gca 105 Arg Ser Leu ArgAla Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala 5 10 15 ccc ctg ttc ctgctg cgc tcc gcc ctg gcc gac ttc agc ctg gac aac 153 Pro Leu Phe Leu LeuArg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn 20 25 30 35 gag gtg cac tcgagc ttc atc cac cgg cgc ctc cgc agc cag gag cgg 201 Glu Val His Ser SerPhe Ile His Arg Arg Leu Arg Ser Gln Glu Arg 40 45 50 cgg gag atg cag cgcgag atc ctc tcc att ttg ggc ttg ccc cac cgc 249 Arg Glu Met Gln Arg GluIle Leu Ser Ile Leu Gly Leu Pro His Arg 55 60 65 ccg cgc ccg cac ctc cagggc aag cac aac tcg gca ccc atg ttc atg 297 Pro Arg Pro His Leu Gln GlyLys His Asn Ser Ala Pro Met Phe Met 70 75 80 ctg gac ctg tac aac gcc atggcg gtg gag gag ggc ggc ggg ccc ggc 345 Leu Asp Leu Tyr Asn Ala Met AlaVal Glu Glu Gly Gly Gly Pro Gly 85 90 95 ggc cag ggc ttc tcc tac ccc tacaag gcc gtc ttc agt acc cag ggc 393 Gly Gln Gly Phe Ser Tyr Pro Tyr LysAla Val Phe Ser Thr Gln Gly 100 105 110 115 ccc cct ctg gcc agc ctg caagat agc cat ttc ctc acc gac gcc gac 441 Pro Pro Leu Ala Ser Leu Gln AspSer His Phe Leu Thr Asp Ala Asp 120 125 130 atg gtc atg agc ttc gtc aacctc gtg gaa cat gac aag gaa ttc ttc 489 Met Val Met Ser Phe Val Asn LeuVal Glu His Asp Lys Glu Phe Phe 135 140 145 cac cca cgc tac cac cat cgagag ttc cgg ttt gat ctt tcc aag atc 537 His Pro Arg Tyr His His Arg GluPhe Arg Phe Asp Leu Ser Lys Ile 150 155 160 cca gaa ggg gaa gct gtc acggca gcc gaa ttc cgg atc tac aag gac 585 Pro Glu Gly Glu Ala Val Thr AlaAla Glu Phe Arg Ile Tyr Lys Asp 165 170 175 tac atc cgg gaa cgc ttc gacaat gag acg ttc cgg atc agc gtt tat 633 Tyr Ile Arg Glu Arg Phe Asp AsnGlu Thr Phe Arg Ile Ser Val Tyr 180 185 190 195 cag gtg ctc cag gag cacttg ggc agg gaa tcg gat ctc ttc ctg ctc 681 Gln Val Leu Gln Glu His LeuGly Arg Glu Ser Asp Leu Phe Leu Leu 200 205 210 gac agc cgt acc ctc tgggcc tcg gag gag ggc tgg ctg gtg ttt gac 729 Asp Ser Arg Thr Leu Trp AlaSer Glu Glu Gly Trp Leu Val Phe Asp 215 220 225 atc aca gcc acc agc aaccac tgg gtg gtc aat ccg cgg cac aac ctg 777 Ile Thr Ala Thr Ser Asn HisTrp Val Val Asn Pro Arg His Asn Leu 230 235 240 ggc ctg cag ctc tcg gtggag acg ctg gat ggg cag agc atc aac ccc 825 Gly Leu Gln Leu Ser Val GluThr Leu Asp Gly Gln Ser Ile Asn Pro 245 250 255 aag ttg gcg ggc ctg attggg cgg cac ggg ccc cag aac aag cag ccc 873 Lys Leu Ala Gly Leu Ile GlyArg His Gly Pro Gln Asn Lys Gln Pro 260 265 270 275 ttc atg gtg gct ttcttc aag gcc acg gag gtc cac ttc cgc agc atc 921 Phe Met Val Ala Phe PheLys Ala Thr Glu Val His Phe Arg Ser Ile 280 285 290 cgg tcc acg ggg agcaaa cag cgc agc cag aac cgc tcc aag acg ccc 969 Arg Ser Thr Gly Ser LysGln Arg Ser Gln Asn Arg Ser Lys Thr Pro 295 300 305 aag aac cag gaa gccctg cgg atg gcc aac gtg gca gag aac agc agc 1017 Lys Asn Gln Glu Ala LeuArg Met Ala Asn Val Ala Glu Asn Ser Ser 310 315 320 agc gac cag agg caggcc tgt aag aag cac gag ctg tat gtc agc ttc 1065 Ser Asp Gln Arg Gln AlaCys Lys Lys His Glu Leu Tyr Val Ser Phe 325 330 335 cga gac ctg ggc tggcag gac tgg atc atc gcg cct gaa ggc tac gcc 1113 Arg Asp Leu Gly Trp GlnAsp Trp Ile Ile Ala Pro Glu Gly Tyr Ala 340 345 350 355 gcc tac tac tgtgag ggg gag tgt gcc ttc cct ctg aac tcc tac atg 1161 Ala Tyr Tyr Cys GluGly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 360 365 370 aac gcc acc aaccac gcc atc gtg cag acg ctg gtc cac ttc atc aac 1209 Asn Ala Thr Asn HisAla Ile Val Gln Thr Leu Val His Phe Ile Asn 375 380 385 ccg gaa acg gtgccc aag ccc tgc tgt gcg ccc acg cag ctc aat gcc 1257 Pro Glu Thr Val ProLys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala 390 395 400 atc tcc gtc ctctac ttc gat gac agc tcc aac gtc atc ctg aag aaa 1305 Ile Ser Val Leu TyrPhe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys 405 410 415 tac aga aac atggtg gtc cgg gcc tgt ggc tgc cac tagctcctcc 1351 Tyr Arg Asn Met Val ValArg Ala Cys Gly Cys His 420 425 430 gagaattcag accctttggg gccaagtttttctggatcct ccattgctcg ccttggccag 1411 gaaccagcag accaactgcc ttttgtgagaccttcccctc cctatcccca actttaaagg 1471 tgtgagagta ttaggaaaca tgagcagcatatggcttttg atcagttttt cagtggcagc 1531 atccaatgaa caagatccta caagctgtgcaggcaaaacc tagcaggaaa aaaaaacaac 1591 gcataaagaa aaatggccgg gccaggtcattggctgggaa gtctcagcca tgcacggact 1651 cgtttccaga ggtaattatg agcgcctaccagccaggcca cccagccgtg ggaggaaggg 1711 ggcgtggcaa ggggtgggca cattggtgtctgtgcgaaag gaaaattgac ccggaagttc 1771 ctgtaataaa tgtcacaata aaacgaatgaatgaaaaaaa aaaaaaaaaa a 1822 2 431 PRT Homo sapiens 2 Met His Val ArgSer Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 15 Leu Trp AlaPro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 20 25 30 Leu Asp AsnGlu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser 35 40 45 Gln Glu ArgArg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu 50 55 60 Pro His ArgPro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro 65 70 75 80 Met PheMet Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly 85 90 95 Gly ProGly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser 100 105 110 ThrGln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr 115 120 125Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys 130 135140 Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 145150 155 160 Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe ArgIle 165 170 175 Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr PheArg Ile 180 185 190 Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg GluSer Asp Leu 195 200 205 Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser GluGlu Gly Trp Leu 210 215 220 Val Phe Asp Ile Thr Ala Thr Ser Asn His TrpVal Val Asn Pro Arg 225 230 235 240 His Asn Leu Gly Leu Gln Leu Ser ValGlu Thr Leu Asp Gly Gln Ser 245 250 255 Ile Asn Pro Lys Leu Ala Gly LeuIle Gly Arg His Gly Pro Gln Asn 260 265 270 Lys Gln Pro Phe Met Val AlaPhe Phe Lys Ala Thr Glu Val His Phe 275 280 285 Arg Ser Ile Arg Ser ThrGly Ser Lys Gln Arg Ser Gln Asn Arg Ser 290 295 300 Lys Thr Pro Lys AsnGln Glu Ala Leu Arg Met Ala Asn Val Ala Glu 305 310 315 320 Asn Ser SerSer Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr 325 330 335 Val SerPhe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu 340 345 350 GlyTyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 355 360 365Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His 370 375380 Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln 385390 395 400 Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn ValIle 405 410 415 Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly CysHis 420 425 430 3 102 PRT Artificial Sequence Description of ArtificialSequence OPX 3 Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu GlyTrp Xaa 1 5 10 15 Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr TyrCys Glu Gly 20 25 30 Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala ThrAsn His Ala 35 40 45 Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa XaaVal Pro Lys 50 55 60 Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser ValLeu Tyr Xaa 65 70 75 80 Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys Xaa ArgAsn Met Val Val 85 90 95 Xaa Ala Cys Gly Cys His 100 4 97 PRT ArtificialSequence Description of Artificial Sequence Generic- Seq-7 4 Leu Xaa XaaXaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Pro XaaXaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro 50 55 60 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 ValXaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys 85 90 95 Xaa5 102 PRT Artificial Sequence Description of Artificial SequenceGeneric- Seq-8 5 Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa GlyTrp Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa TyrCys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaAsn His Ala 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 50 55 60 Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaLeu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa XaaXaa Met Xaa Val 85 90 95 Xaa Xaa Cys Xaa Cys Xaa 100 6 97 PRT ArtificialSequence Description of Artificial Sequence Generic- Seq-9 6 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Pro XaaXaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro 50 55 60 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 85 90 95 Xaa7 102 PRT Artificial Sequence Description of Artificial SequenceGeneric- Seq-10 7 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa XaaXaa Cys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Leu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Cys Xaa Cys Xaa 100 8 5 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide8 Cys Xaa Xaa Xaa Xaa 1 5 9 5 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 9 Cys Xaa Xaa Xaa Xaa 1 5 10 102PRT Artificial Sequence Description of Artificial Sequence Generic aminoacid sequence 10 Cys Xaa Xaa Xaa Xaa Leu Xaa Val Xaa Phe Xaa Asp Xaa GlyTrp Xaa 1 5 10 15 Xaa Trp Xaa Xaa Xaa Pro Xaa Gly Xaa Xaa Ala Xaa TyrCys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaAsn His Ala 35 40 45 Xaa Xaa Gln Xaa Xaa Val Xaa Xaa Xaa Asn Xaa Xaa XaaXaa Pro Xaa 50 55 60 Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaLeu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Tyr XaaXaa Met Xaa Val 85 90 95 Xaa Xaa Cys Xaa Cys Xaa 100 11 96 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid (COP5) 11 Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp Asp Trp IleVal Ala 1 5 10 15 Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His Gly Glu CysPro Phe Pro 20 25 30 Leu Ala Asp His Phe Asn Ser Thr Asn His Ala Val ValGln Thr Leu 35 40 45 Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys CysVal Pro Thr 50 55 60 Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu AsnGlu Lys Val 65 70 75 80 Val Leu Lys Tyr Asn Gln Glu Met Val Val Glu GlyCys Gly Cys Arg 85 90 95 12 96 PRT Artificial Sequence Description ofArtificial Sequence Synthetic amino acid (COP7) 12 Leu Tyr Val Asp PheSer Asp Val Gly Trp Asn Asp Trp Ile Val Ala 1 5 10 15 Pro Pro Gly TyrHis Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro 20 25 30 Leu Ala Asp HisLeu Asn Ser Thr Asn His Ala Val Val Gln Thr Leu 35 40 45 Val Asn Ser ValAsn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr 50 55 60 Glu Leu Ser AlaIle Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val 65 70 75 80 Val Leu LysTyr Asn Gln Glu Met Val Val Glu Gly Cys Gly Cys Arg 85 90 95 13 97 PRTArtificial Sequence Description of Artificial Sequence Generic aminoacid sequence 13 Leu Xaa Val Xaa Phe Xaa Asp Xaa Gly Trp Xaa Xaa Trp XaaXaa Xaa 1 5 10 15 Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa CysXaa Xaa Pro 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa XaaGln Xaa Xaa 35 40 45 Val Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Pro Xaa Xaa CysCys Val Pro 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa XaaXaa Xaa Xaa 65 70 75 80 Val Xaa Leu Xaa Xaa Tyr Xaa Xaa Met Xaa Val XaaXaa Cys Xaa Cys 85 90 95 Xaa 14 102 PRT Artificial Sequence Descriptionof Artificial Sequence Generic amino acid sequence 14 Cys Xaa Arg XaaXaa Leu Xaa Val Xaa Phe Xaa Asp Xaa Gly Trp Xaa 1 5 10 15 Xaa Trp XaaXaa Xaa Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 20 25 30 Xaa Cys XaaXaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala 35 40 45 Xaa Xaa GlnXaa Xaa Val Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Pro Xaa 50 55 60 Xaa Cys CysVal Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa XaaXaa Xaa Xaa Val Xaa Leu Xaa Xaa Tyr Xaa Xaa Met Xaa Val 85 90 95 Xaa XaaCys Xaa Cys Xaa 100

What is claimed is:
 1. A method for improving the tissue inductivecapability of a morphogenic protein at a target locus in a mammal, themethod comprising administering to the target locus the morphogenicprotein and a first effective amount of a hormone and a second effectiveamount of a soluble receptor of the hormone, wherein the morphogenicprotein is capable of inducing tissue formation when accessible to aprogenitor cell in the mammal, and the hormone and the receptor incombination enhance that capability.
 2. The method of claim 1, whereinthe morphogenic protein comprises a pair of subunits disulfide-bonded toproduce a dimeric species, wherein at least one of the subunitscomprises a polypeptide belonging to the BMP protein family.
 3. Themethod of claim 1, wherein the morphogenic protein is an osteogenicprotein.
 4. The method of claim 3, wherein the osteogenic protein iscapable of inducing the progenitor cell to form endochondral orintramembranous bone.
 5. The method of claim 3, wherein the osteogenicprotein is capable of inducing the progenitor cell to form cartilage. 6.The method of claim 1, wherein the morphogenic protein is capable ofinducing the progenitor cell to form tissue tendon/ligament-like orneural-like tissue.
 7. The method of claim 1, wherein the morphogenicprotein comprises an amino acid sequence sufficiently duplicative of theamino acid sequence of BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7 (OP-1),BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, COP-5, or COP-7.
 8. Themethod of claim 7, wherein the morphogenic protein comprises apolypeptide selected from the group consisting of OP-1, BMP-2, BMP-4 andBMP-6.
 9. The method of claim 8, wherein the morphogenic protein isOP-1.
 10. The method of claim 2, wherein the dimer is a homo- orheterodimer comprising a BMP-2 or BMP-7 (OP-1) subunit.
 11. The methodof claim 1, wherein the hormone is interleukin 6 (IL-6).
 12. The methodof claim 7, wherein the hormone is IL-6.
 13. The method of claim 9,wherein the hormone is IL-6.
 14. The method of claim 1, wherein themorphogenic protein, the hormone, and the hormone receptor areadministered simultaneously to the target locus.
 15. The method of claim1, wherein the morphogenic protein, the hormone, and the hormonereceptor are administered separately to the target locus.
 16. The methodof claim 1, wherein the hormone and the hormone receptor areadministered simultaneously to the target locus.
 17. The method of claim1, wherein the target locus is a jaw bone defect.
 18. The method ofclaim 1, wherein the target locus is a bone defect selected from thegroup consisting of a fracture, a non-union fracture, a critical sizedefect, a non-critical size defect, an osteochondral defect, a fusionand a bony void.
 19. The method of claim 1, wherein the target locus hasa tissue degenerative condition.
 20. The method of claim 1, wherein thetarget locus is a cartilage or soft tissue defect.
 21. The method ofclaim 1, wherein the target locus is a neural tissue defect.
 22. Themethod of claim 1, wherein the morphogenic protein is administered in amatrix-comprising carrier.
 23. The method of claim 22, wherein thecarrier comprises allogenic bone.
 24. The method of claim 1, wherein themorphogenic protein is administered via a nucleic acid, the nucleic acidcomprising a sequence encoding the morphogenic protein and capable ofexpressing the morphogenic protein in the progenitor cell.
 25. Apharmaceutical composition for inducing tissue formation in a mammal,comprising a morphogenic protein, a hormone and a soluble receptor ofthe hormone, wherein the morphogenic protein is capable of inducingtissue formation when accessible to a progenitor cell in the mammal, andthe hormone and the receptor in combination enhance that capability. 26.The pharmaceutical composition of claim 25, further comprising animplantable, biocompatible carrier.
 27. The pharmaceutical compositionof claim 26, wherein the carrier comprises demineralized,protein-extracted, particulate, allogenic bone.
 28. The pharmaceuticalcomposition of claim 26, wherein the carrier comprises mineral-free,delipidated Type I insoluble bone collagen particles, substantiallydepleted in noncollagenous protein.
 29. The pharmaceutical compositionof claim 25, wherein the morphogenic protein comprises an amino acidsequence sufficiently duplicative of the amino acid sequence of BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-7 (OP-1), BMP-8, BMP-9, BMP-10, BMP-11,BMP-12, BMP-13, COP-5, or COP-7.
 30. The pharmaceutical composition ofclaim 29, wherein the morphogenic protein is human OP-1.
 31. Thepharmaceutical composition of claim 25, wherein the hormone is IL-6. 32.A kit for inducing tissue formation in a mammal, comprising a firstreceptacle containing a morphogenic protein, a second receptaclecontaining a hormone, and a third receptacle containing a solublereceptor of the hormone, wherein the morphogenic protein is capable ofinducing tissue formation when accessible to a progenitor cell in themammal, and the hormone and the receptor in combination enhance thatcapability.
 33. The kit of claim 32, wherein the second and the thirdreceptacles are the same.